Nucleic acid probes and methods for detecting Enterobacter cloacae

ABSTRACT

A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed.

The present application is a divisional of Hogan et al., U.S. application Ser. No. 08/200,866, filed Feb. 22, 1994, now U.S. Pat. No. 5,541,308 which is file wrapper continuation of Hogan et al., U.S. application Ser. No. 07/806,929, filed Dec. 11, 1991, now abandoned, which is a file wrapper continuation of Hogan et al., U.S. Ser. No. 07/295,208, filed Dec. 9, 1988, now abandoned, which was the National filing of PCT/US87/03009, filed Nov. 24, 1987, which is a continuation-in-part of Hogan et al., U.S. application Ser. No. 07/083,542, filed Aug. 7, 1987, now abandoned, which is a continuation-in-part of Hogan et al., U.S. Ser. No. 06/934,244, filed Nov. 24, 1986, now abandoned, the entirely of each of these prior applications including drawings are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The inventions described and claimed herein relate to probes and assays based on the use of genetic material such as RNA. More particularly, the inventions relate to the design and construction of nucleic acid probes and hybridization of such probes to genetic material of target non-viral organisms in assays for detection and/or quantitation thereof in test samples of, e.g., sputum, urine, blood and tissue sections, food, soil and water.

2. Introduction

Two single strands of nucleic acid, comprised of nucleotides, may associate ("hybridize") to form a double helical structure in which the two polynucleotide chains running in opposite directions are held together by hydrogen bonds (a weak form of chemical bond) between pairs of matched, centrally located compounds known as "bases." Generally, in the double helical structure of nucleic acids, for example, the base adenine (A) is hydrogen bonded to the base thymine (T) or uracil (U) while the base guanine (G) is hydrogen bonded to the base cytosine (C). At any point along the chain, therefore, one may find the base pairs AT or AU, TA or UA, GC, or CG. One may also find AG and GU base pairs in addition to the traditional ("canonical") base pairs. Assuming that a first single strand of nucleic acid is sufficiently complementary to a second and that the two are brought together under conditions which will promote their hybridization, double stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed.

Broadly, there are two basic nucleic acid hybridization procedures. In one, known as "in solution" hybridization, both a "probe" nucleic acid sequence and nucleic acid molecules from a test sample are free in solution. In the other method, the sample nucleic acid is usually immobilized on a solid support and the probe sequence is free in solution.

A probe may be a single strand nucleic acid sequence which is complementary in some particular degree to the nucleic acid sequences sought to be detected ("target sequences"). It may also be labelled. A background description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is described in U.S. application Ser. No. 456,729, entitled "Method for Detection, Identification and Quantitation of Non-Viral Organisms," filed Jan. 10, 1983 (Kohne I, now issued as U.S. Pat. No. 4,851,330), and U.S. application Ser. No. 655,365, entitled "Method For Detecting, Identifying and Quantitating organisms and Viruses," filed Sep. 4, 1984 (Kohne II, now issued as U.S. Pat. No. 5,288,611) both of which are incorporated by reference, together with all other applications cited herein.

Also described in those applications are methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms, comprising the steps of bringing together any nucleic acids from a sample and a probe comprising nucleic acid molecules which are shorter than the rRNA subunit sequence from which it was derived and which are sufficiently complementary to hybridize to the rRNA of one or more non-viral organisms or groups of non-viral organisms, incubating the mixture under specified hybridization conditions, and assaying the resulting mixture for hybridization of the probe and any test sample rRNA. The invention is described to include using a probe which detects only rRNA subunit subsequences which are the same or sufficiently similar in particular organisms or groups of organisms and is said to detect the presence or absence of any one or more of those particular organisms in a sample, even in the presence of many non-related organisms.

We have discovered and describe herein a novel method and means for designing and constructing DNA probes for use in detecting unique rRNA sequences in an assay for the detection and/or quantitation of any group of non-viral organisms. Some of the inventive probes herein may be used to detect and/or quantify a single species or strain of non-viral organism and others may be used to detect and/or quantify members of an entire genus or desired phylogenetic grouping.

SUMMARY OF THE INVENTION

In a method of probe preparation and use, a single strand deoxyoligonucleotide of particular sequence and defined length is used in a hybridization assay to determine the presence or amount of rRNA from particular target non-viral organisms to distinguish them from their known closest phylogenetic neighbors. Probe sequences which are specific, respectively, for 16S rRNA variable subsequences of Mycobacterium avium, Mycobacterium intracellulare and the Mycobacterium tuberculosis-complex bacteria, and which do not cross react with nucleic acids from each other, or any other bacterial species or respiratory infectious agent, under proper stringency, are described and claimed. A probe specific to three 23S rRNA variable region subsequences from the Mycobacterium tuberculosis-complex bacteria is also described and claimed, as are rRNA variable region probes useful in hybridization assays for the genus Mycobacterium (16S 23S rRNA specific), Mycoplasma pneumoniae (5S and 16S rRNA-specific), Chlamydia trachomatis (16S and 23S rRNA specific), Enterobacter cloacae (23S rRNA specific), Escherichia coli (16S rRNA specific), Legionella (16S and 23S rRNA specific), Salmonella (16S and 23S rRNA specific), Enterococci (16S rRNA specific), Neisseria gonorrhoeae (16S rRNA specific), Campylobacter (16S rRNA specific), Proteus mirabilis (23S rRNA specific), Pseudomonas (23S rRNA specific), fungi (18S and 28S rRNA specific), and bacteria (16S and 23S rRNA specific).

In one embodiment of the assay method, a test sample is first subjected to conditions which release rRNA from any non-viral organisms present in that sample. rRNA is single stranded and therefore available for hybridization with sufficiently complementary genetic material once so released. Contact between a probe, which can be labelled, and the rRNA target may be carried out in solution under conditions which promote hybridization between the two strands. The reaction mixture is then assayed for the presence of hybridized probe. Numerous advantages of the present method for the detection of non-viral organisms over prior art techniques, including accuracy, simplicity, economy and speed will appear more fully from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1, 1A and 1B is a chart of the primary structure of bacterial 16S rRNA for Escherichia coli, depicting standard reference numbers for bases.

FIGS. 2, 2A, 2B, 2C, and 2D is a chart of the primary structure of bacterial 23S rRNA for Escherichia coli, depicting standard reference numbers for bases.

FIGS. 3, 3A, and 3B is a chart of the primary structure of bacterial 5S rRNA for Escherichia coli, depicting standard reference numbers for bases.

FIGS. 4, 4A, 4B, and 4C is a chart of the primary structure for the 18S rRNA for Saccharomyces cerevisiae, depicting standard reference numbers for bases.

FIGS. 5, 5A, 5B, 5C, 5D, 5E, and 5F is a chart of the primary structure for the 28S rRNA for Saccharomyces cerevisiae, depicting standard reference numbers for bases.

FIG. 6 is a diagram showing the locations in the 16S rRNA (using E. coli reference numbers) which differ between 12 different sets of related organisms. In Example 1, for example, 99.7% refers to the difference in 16s rRNA between Clostridium botuliniuma and Clostridium subterminale.

FIG. 7 is a diagram showing the locations in the first 1500 bases of 23S rRNA (using E. coli reference numbers) which differ between 12 different sets of related organisms.

FIG. 8 is a diagram showing the locations in the terminal bases of 23S rRNA (using E. coli reference numbers) which differ between 12 different sets of related organisms.

FIG. 9 is a schematic representation of the location of probes capable of hybridizing to the 16S rRNA.

FIG. 10 is a schematic representation of the location of probes capable of hybridizing to the first 1500 bases of the 23S rRNA.

FIG. 11 is a schematic representation of the location of probes capable of hybridizing to the terminal bases of 23S rRNA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms, as used in this disclosure and claims, are defined as:

nucleotide: a subunit of a nucleic acid consisting of a phosphate group, a 5' carbon sugar and a nitrogen containing base. In RNA the 5' carbon sugar is ribose. In DNA, it is a 2-deoxyribose. The term also includes analogs of such subunits.

nucleotide polymer: at least two nucleotides linked by phosphodiester bonds.

oligonucleotide: a nucleotide polymer generally about 10 to about 100 nucleotides in length, but which may be greater than 100 nucleotides in length.

nucleic acid probe: a single stranded nucleic acid sequence that will combine with a complementary single stranded target nucleic acid sequence to form a double-stranded molecule (hybrid). A nucleic acid probe may be an oligonucleotide or a nucleotide polymer.

hybrid: the complex formed between two single stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases.

hybridization: the process by which two complementary strands of nucleic acids combine to form double stranded molecules (hybrids).

complementarity: a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) usually complements thymine (T) or Uracil (U), while guanine (G) usually complements cytosine (C).

stringency: term used to describe the temperature and solvent composition existing during hybridization and the subsequent processing steps. Under high stringency conditions only highly homologous nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determine the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency is chosen to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid.

probe specificity: characteristic of a probe which describes its ability to distinguish between target and non-target sequences. Dependent on sequence and assay conditions. Probe specificity may be absolute (i.e., probe able to distinguish between target organisms and any nontarget organisms), or it may be functional (i.e., probe able to distinguish between the target organism and any other organism normally present in a particular sample). Many probe sequences can be used for either broad or narrow specificity depending on the conditions of use.

variable region: nucleotide polymer which differs by at least one base between the target organism and nontarget organisms contained in a sample.

conserved region: a region which is not variable.

sequence divergence: process by which nucleotide polymers become less similar during evolution.

sequence convergence: process by which nucleotide polymers become more similar during evolution.

bacteria: members of the phylogenetic group eubacteria, which is considered one of the three primary kingdoms.

Tm: temperature at which 50% of the probe is converted from the hybridized to the unhybridized form.

thermal stability: Temperature at which 50% of the probe:target hybrids are converted to the single stranded form. Factors which affect the thermal stability can affect probe specificity and therefore, must be controlled. Whether a probe sequence is useful to detect only a specific type of organism depends largely on the thermal stability difference between probe:target hybrids ("P:T") and probe:nontarget hybrids ("P:NT"). In designing probes the Tm P:T minus the Tm P:NT should be as large as possible.

In addition to a novel method for selecting probe sequences, we have discovered that it is possible to create a DNA probe complementary to a particular rRNA sequence obtained from a single type of target microorganism and to successfully use that probe in a non-cross reacting assay for the detection of that single microorganism, even in the presence of its known, most closely related taxonomic or phylogenetic neighbors. With the exception of viruses, all prokaryotic organisms contain rRNA molecules including 5S rRNA, 16S rRNA, and a larger rRNA molecule known as 23S rRNA. Eukaryotes are known to have 5.0S, 5.8S, 18S and 28S rRHA molecules or analogous structures. (The term "16S like" sometimes is used to refer to the rRNA found in the small ribosomal subunit, including 18S and 17S rRNA. Likewise the term "23S like" rRNA sometimes is used to refer to the rRNA found in the large ribosomal subunit. 5.8S rRNA is equivalent to the 5' end of the 23S like rRNA.) These rRNA molecules contain nucleotide sequences which are highly conserved among all organisms thus far examined. There are known methods which allow a significant portion of these rRNA sequences to be determined. For example, complementary oligonucleotide primers of about 20-30 bases in length can be hybridized to universally conserved regions in purified rRNA that are specific to the 5S, 16S, or 23S subunits and extended with the enzyme reverse transcriptase. Chemical degradation or dideoxynucleotide- terminated sequencing reactions can be used to determine the nucleotide sequence of the extended product. Lane, D. J. et al., Proc. Nat'l Acad. Sci. USA 82, 6955-6959 (1985).

In our invention, comparison of one or more sequenced rRNA variable regions from a target organism to one or more rRNA variable region sequences from a closely related bacterial species is utilized to select a sequence unique to the rRNA of the target organism. rRNA is preferable to DNA as a probe target because of its relative abundance and stability in the cell and because of its patterns of phylogenetic conservation.

Notwithstanding the highly conserved nature of rRNA, we have discovered that a number of regions of the rRNA molecule which can vary in sequence, can vary even between closely related species and can, therefore, be utilized to distinguish between such organisms. Differences in the rRNA molecule are not distributed randomly across the entire molecule, but rather are clustered into specific regions. The degree of conservation also varies, creating a unique pattern of conservation across the ribosomal RNA subunits. The degree of variation and the distribution thereof, can be analyzed to locate target sites for diagnostic probes. This method of probe selection may be used to select more than one sequence which is unique to the rRNA of a target organism.

We have identified variable regions by comparative analysis of rRNA sequences both published in the literature and sequences which we have determined ourselves using procedures known in the art. We use a Sun Microsystems (TM) computer for comparative analysis. The compiler is capable of manipulating many sequences of data at the same time. Computers of this type and computer programs which may be used or adapted for the purposes herein disclosed are commercially available.

Generally, only a few regions are useful for distinguishing between closely related species of a phylogenetically conserved genus, for example, the region 400-500 bases from the 5' end of the 16S rRNA molecule. An analysis of closely related organisms (FIGS. 6, 7 and 8) reveals the specific positions (variable regions) which vary between closely related organisms. These variable regions of rRNA molecules are the likely candidates for probe design.

FIGS. 6, 7 and 8 display the variations in 16S and 23S rRNA's between two different bacteria with decreasing amounts of similarity between them. Closer analysis of these figures reveals some subtle patterns between these closely related organisms. In all cases studied, we have seen sufficient variation between the target organism and the closest phylogenetic relative found in the same sample to design the probe of interest. Moreover, in all cases studied to date, the per cent similarity between the target organism (or organisms) and the closest phylogenetically related organisms found in the same sample has been between 90% and 99%. Interestingly, there was enough variation even between the rRNA's of Neisseria's gonorrhoeae and meningitidis (See Example 21) to design probes--despite the fact that DNA:DNA homology studies suggested these two species might actually be one and the same.

These figures also show that the differences are distributed across the entire 16S and 23S rRNA's. Many of the differences, nonetheless, cluster into a few regions. These locations in the rRNA are good candidates for probe design, with our current assay conditions. We also note that the locations of these increased variation densities usually are situated in the same regions of the 16S and 23S rRNA for comparable per cent similarity values. In this manner, we have observed that certain regions of the 16S and 23S rRNA are the most likely sites in which significant variation exists between the target organism and the closest phylogenetic relatives found in a sample. We have disclosed and claimed species specific probes which hybridize in these regions of significant variation between the target organism and the closest phylogenetic relative found in a sample.

FIGS. 9, 10 and 11 are a schematic representation of the location of probes disclosed and claimed herein. Because 16S and 23S RNAs do not, as a rule, contain sequences of duplication longer than about six nucleotides in length, probes designed by these methods are specific to one or a few positions on the target nucleic acid.

The sequence evolution at each of the variable regions (for example, spanning a minimum of 10 nucleotides) is, for the most part divergent, not convergent. Thus, we can confidently design probes based on a few rRNA sequences which differ between the target organism and its phylogenetically closest relatives. Biological and structural constraints on the rRNA molecule which maintain homologous primary, secondary and tertiary structure throughout evolution, and the application of such constraints to probe diagnostics is the subject of ongoing study. The greater the evolutionary distance between organisms, the greater the number of variable regions which may be used to distinguish the organisms.

Once the variable regions are identified, the sequences are aligned to reveal areas of maximum homology or "match". At this point, the sequences are examined to identify potential probe regions. Two important objectives in designing a probe are to maximize homology to the target sequence(s) (greater than 90% homology is recommended) and to minimize homology to non-target sequence(s) (less than 90% homology to nontargets is recommended). We have identified the following useful guidelines for designing probes with desired characteristics.

First, probes should be positioned so as to minimize the stability of the probe:nontarget nucleic acid hybrid. This may be accomplished by minimizing the length of perfect complementarity to non-target organisms, avoiding G and C rich regions of homology to non-target sequences, and by positioning the probe to span as many destabalizing mismatches as possible (for example, dG:rU base pairs are less destabalizing than some others).

Second, the stability of the probe:target nucleic acid hybrid should be maximized. This may be accomplished by avoiding long A and T rich sequences, by terminating the hybrids with G:C base pairs and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and %G and %C result in a Tm about 2-10° C. higher than the temperature at which the final assay will be performed. The importance and effect of various assay conditions will be explained further herein. Third, regions of the rRNA which are known to form strong structures inhibitory to hybridization are less preferred. Finally, probes with extensive self-complementarity should be avoided.

In some cases, there may be several sequences from a particular region which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base.

The following chart indicates how, for one embodiment of the invention useful in the detection of a nucleic acid in the presence of closely related nucleic acid sequences, unique sequences can be selected. In this example, rRNA sequences have been determined for organisms A-E and their sequences, represented numerically, are aligned as shown. It is seen that sequence 1 is common to all organisms A-E. Sequences 2-6 are found only in organisms A, B and C, while sequences 8, 9 and 10 are unique to organinm A. Therefore, a probe complementary to sequences 8, 9 or 10 would specifically hybridize to organism A.

    ______________________________________     Illustrative Pattern of Sequence     Relationships Among Releated Bacteria     Organism             rRNA Sequence     ______________________________________     A       1     2      3   4    5   6    7   8    9   10     B       1     2      3   4    5   6    7   11   12  13     C       1     2      3   4    5   6    14  15   16  17     D       1     18     19  20   21  22   23  24   25  26     E       1     18     19  20   21  27   28  29   30  31     ______________________________________

In cases where the patterns of variation of a macromolecule are known, for example, rRNA, one can focus on specific regions as likely candidates for probe design. However, it is not always necessary to determine the entire nucleic acid sequence in order to obtain a probe sequence. Extension from any single oligonucleotide primer can yield up to 300-400 bases of sequence. When a single primer is used to partially sequence the rRNA of the target organism and organisms closely related to the target, an alignment can be made as outlined above. Plainly, if a useful probe sequence is found, it is not necessary to continue rRNA sequencing using other primers. If, on the other hand, no useful probe sequence is obtained from sequencing with a first primer, or if higher sensitivity is desired, other primers can be used to obtain more sequences. In those cases where patterns of variation for a molecule are not well understood, more sequence data may be required prior to probe design.

Thus, in Examples 1-3 below, two 16S-derived primers were used. The first primer did not yield probe sequences which met the criteria listed herein. The second primer yielded probe sequences which were determined to be useful following characterization and testing for specificity as described. In Example 4, six 23S primers were used prior to locating the probe sequence set forth.

Once a presumptive unique sequence has been identified, a complementary DNA oligonucleotide is synthesized. This single stranded oligonucleotide will serve as the probe in the DNA/rRNA assay hybridization reaction. Defined oligonucleotides may be synthesized by any of several well known methods, including automated solid-phase chemical synthesis using cyano-ethylphosphoramidite precursors. Barone, A. D. et al., Nucleic Acids Research 12, 4051-4060 (1984). In this method, deoxyoligonucleotides are synthesized on solid polymer supports. Release of the oligonucleotide from the support is accomplished by treatment with ammonium hydroxide at 60° C. for 16 hours. The solution is dried and the crude product is dissolved in water and separated on polyacrylamide gels which generally may vary from 10-20% depending upon the length of the fragment. The major band, which is visualized by ultraviolet back lighting, is cut from the gel with a razor blade and extracted with 0.1 M ammonium acetate, pH 7.0, at room temperature for 8-12 hours. Following centrifugation, the supernatant is filtered through a 0.4 micron filter and desalted on a P-10 column (Pharmacia). Other well known methods for construction of synthetic oligonucelotides may, of course, be employed.

Current DNA synthesizers can produce large amounts of synthetic DNA. After synthesis, the size of the newly made DNA is examined by gel filtration and molecules of varying size are generally detected. Some of these molecules represent abortive synthesis events which occur during the synthesis process. As part of post-synthesis purification, the synthetic DNA is usually size fractionated and only those molecules which are the proper length are kept. Thus, it is possible to obtain a population of synthetic DNA molecules of uniform size.

It has been generally assumed, however, that synthetic DNA is inherently composed of a uniform population of molecules all of the same size and base sequence, and that the hybridization characteristics of every molecule in the preparation should be the same. In reality, preparations of synthetic DNA molecules are heterogeneous and are composed of significant numbers of molecules which, although the same size, are in some way different from each other and have different hybridization characteristics. Even different preparations of the same sequence can sometimes have different hybridization characteristics.

Accordingly, preparations of the same synthetic probe sequence can have different hybridization chacteristics. Because of this the specificity of probe molecules from different preparations can be different. The hybridization characteristics of each preparation should be examined in order to determine the hybridization conditions which must be used in order to obtain the desired probe specificity. For example, the synthetic probe described in Example 4 below has the specificity profile described in Table 14. This data was obtained by using the hybridization and assay conditions described. A separate preparation of this probe which has different hybridization characteristics may not have precisely the same specificity profile when assayed under the conditions presented in Example 4. Such probe preparations have been made. To obtain the desired specificity, these probes can be hybridized and assayed under different conditions, including salt concentration and/or temperature. The actual conditions under which the probe is to be used must be determined, or matched to extant requirements, for each batch of probe since the art of DNA synthesis is somewhat imperfect.

Following synthesis and purification of a particular oligonucleotide sequence, several procedures may be utilized to determine the acceptability of the final product. The first is polyacrylamide gel electrophoresis, which is used to determine size. The oligonucleotide is labelled using, for example, ³² P-ATP and T₄ polynucleotide kinase. The labelled probe is precipitated in ethanol, centrifuged and the dried pellet resuspended in loading buffer (80% formamide, 20 mM NaOH, 1 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol). The samples are heated for five minutes at 90° C. and loaded onto a denaturing polyacrylamide gel. Electrophoresis is carried out in TBE buffer (0.1 M Tris HCl pH 8.3, 0.08 M boric acid, 0.002 M EDTA) for 1-2 hours at 1,000 volts. Following electrophoresis of the oligonucleotide the gel is exposed to X-ray film. The size of the oligonucleotide is then computed from the migration of oligonucleotide standards run concurrently.

The sequence of the synthetic oligonucleotide may also be checked by labelling it at the 5' end with ³² P-ATP and T₄ polynucleotide kinase, subjecting it to standard chemical degradation techniques, Maxam, A. M. and Gilbert, W., Proc. Nat'l. Acad. Sci, USA 74, 560-564 (1980), and analyzing the products on polyacrylamide gels. Preferably, the nucleotide sequence of the probe is perfectly complementary to the previously identified unique rRNA sequence, although it need not be.

The melting profile, including the melting temperature (Tm) of the oligonucleotide/rRNA hybrids should also be determined. One way to determine Tm is to hybridize a ³² P-labelled oligonucleotide to its complementary target nucleic acid at 50° C. in 0.1 M phosphate buffer, pH 6.8. The hybridization mixture is diluted and passed over a 2 cm hydroxyapatite column at 50° C. The column is washed with 0.1 M phosphate buffer, 0.02% SDS to elute all unhybridized, single-stranded probes. The column temperature is then dropped 15° C. and increased in 5° C. increments until all of the probe is single-stranded. At each temperature, unhybridized probe is eluted and the counts per minute (cpm) in each fraction determined. The number of cpm shown to be bound to the hydroxyapatite divided by the total cpm added to the column equals the percent hybridization of the probe to the target nucleic acid.

An alternate method for determining thermal stability of a hybrid is outlined below. An aliquot of hybrid nucleic acid is diluted into 1 ml of either 0.12 M phosphate buffer, 0.2% SDS, 1 mM EDTA, 1 mM EGTA or an appropriate hybridization buffer. Heat this 1 ml of solution to 45° C. for 5 minutes and place it into a room temperature water bath to cool for 5 minutes. Assay this 1 ml of hybrid containing solution over a hydroxyapatite column, capturing the hybrid and washing away unbound probe. If a hybridization solution other than the 0.12M phosphate buffer is used, then a dilution of the hybridization solution into the 0.12 M phosphate buffer will be necessary for binding. Keep taking aliquots of hybrid and diluting into 1 ml of hybridization solution or into the standard 0.12 M phosphate buffer solution described above while raising the heating temperature 5° C. at a time. Continue this until all of the hybrid is dissociated. The point where one half of the hybrid is converted to the dissociated form is considered the Tm. The Tm for a given hybrid will vary depending on the hybridization solution being used because the thermal stability depends upon the concentration of different salts, detergents, and other solutes which effect relative hybrid stability during thermal denaturation.

Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. For example, the base composition of the probe may be significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be stable at higher temperatures.

We have discovered that the length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly homologous base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, oligonucleotide probes preferred in this invention are between about 15 and about 50 bases in length and are at least about 75-100% homologous to the target nucleic acid. For most applications 95-100% homology to the target nucleic acid is preferred.

Ionic strength and incubation temperature should also be taken into account in constructing a probe. It is known that the rate of hybridization will increase as ionic strength of the reaction mixture increases and that the thermal stability of hybrids will increase with increasing ionic strength. In general, optimal hybridization for synthetic oligonucleotide probes of about 15-50 bases in length occurs approximately 5° C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.

As to nucleic acid concentration, it is known that the rate of hybridization is proportional to the concentration of the two interacting nucleic acid species. Thus, the presence of compounds such as dextran and dextran sulphate are thought to increase the local concentration of nucleic acid species and thereby result in an increased rate of hybridization. Other agents which will result in increased rates of hybridization are specified in U.S. application Ser. No. 627,795, entitled "Accelerated Nucleic Acid Reassociation Method", filed Jul. 5, 1984, continuation-in-Part thereof, Ser. No. (net yet assigned), filed Jun. 4, 1987, and U.S. application Ser. No. 816,711, entitled "Accelerated Nucleic Acid Reassociation Method", filed Jan. 7, 1986, both of which are incorporated by reference. (U.S. application Ser. No. 07/644,879, which is a continuation of U.S. application Ser. No. 816,711, issued as U.S. Pat. No. 5,132,207, on Jul. 21, 1992.) On the other hand, chemical reagents which disrupt hydrogen bonds such as formamide, urea, DMSO, and alcohols will increase the stringency of hybridization.

Selected oligonucleotide probes may be labelled by any of several well known methods. Useful labels include radioisotopes as well as non-radioactive reporting groups. Isotopic labels include ³ H, ³⁵ S, ³² P, ¹²⁵ I, Cobalt and ¹⁴ C. Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription. When using radio-labelled probes, hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling.

Non-isotopic materials can also be used for labelling, and may be introduced by the incorporation of modified nucleotides through the use of enzymes or by chemical modification of the probe, for example, by the use of non-nucleotide linker groups. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands. We currently prefer to use acridinium esters.

In one embodiment of the DNA/rRNA hybridization assay invention, a labelled probe and bacterial target nucleic acids are reacted in solution. rRNA may be released from bacterial cells by the sonic disruption method described in Murphy, K. A. et al., U.S. application Ser. No. 841,860, entitled "Method for Releasing RNA and DNA From Cells", filed Mar. 20, 1986, which is incorporated herein by reference. (U.S. application Ser. No. 07/711,114, which is a continuation of U.S. application Ser. No. 07/298,765, which is a continuation of U.S. application Ser. No. 06/841,860, issued as U.S. Pat. No. 5,374,522, on Jan. 20, 1994.) Other known methods for disrupting cells include the use of enzymes, osmotic shock, chemical treatment, and vortexing with glass beads. Following or concurrent with the release of rRNA, labelled probe may be added in the presence of accelerating agents and incubated at the optimal hybridization temperature for a period of time necessary to achieve significant reaction. Following this incubation period, hydroxyapatite may be added to the reaction mixture to separate the probe/rRNA hybrids from the non-hybridized probe molecules. The hydroxyapatite pellet is washed, recentrifuged and hybrids detected by means according to the label used.

Twenty-one embodiments illustrative of the claimed inventions are set forth below, in which a synthetic probe or probes complementary to a unique rRNA sequence from a target organism, or group of organisms is determined, constructed and used in a hybridization assay.

DESCRIPTION OF PARTICULAR EMBODIMENTS

Mycobacterium are acid-fast, alcohol fast, aerobic, non-mobile bacilli. Their lipid content is high and their growth slow. Mycobacterium avium and Mycobacterium intracellulare are together referred to as M. avium-intracellulare because they are so difficult to differentiate. Recently, the M. avium complex, which includes M. intracellulare, was shown to be the second most commonly isolated, clinically significant Mycobacterium. Good, R. C. et al., J. Infect. Dis. 146, 829-833 (1982). More recent evidence indicates that these organisms are a common cause of opportunistic infection in patients with AIDS (acquired immune deficiency syndrome). Gill, V. J. et al., J. Clin. Microbio. 22, 543-546 (1985). Treatment of such infections in AIDS patients is difficult because these organisms are resistant to most antituberculosis drugs. Often a combination of five drugs are used in therapy. The severity of these infections also requires rapid diagnosis which, prior to the invention herein, was not available.

Members of the Mycobacterium tuberculosis complex (Mtb) include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium microti. The first three are pathogenic for humans while the last is an animal pathogen. These organisms produce slowly developing granulomas on the skin or they may invade internal organs. Tuberculosis of the lungs can be disseminated to other parts of the body by the circulatory system, the lymph system, or the intestinal tract. Despite advances in public health and the advent of effective chemotherapy, Mycobacterial disease, tuberculosis in particular, continues to represent a major world-wide health problem.

The classical method for detecting bacteria in a test sample involves culturing of the sample in order to expand the number of bacterial cells present into observable colony growths which can be identified and enumerated. If desired, the cultures can also be subjected to additional testing in order to determine antimicrobial susceptibility. Currently, the most widely used procedures for the detection, isolation and identification of Mycobacterium species are the acid-fast bacilli (AFB) smear (using either the Ziehl-Neelsen or fluorochrome techniques), culture methods using Lowenstein-Jensen media and Middlebrook media, and biochemical tests. The AFB relies on the high lipid content of Mycobacterium to retain dye after exposure to acid-alcohol. While the AFB smear test is relatively rapid and simple to perform it does not always detect Mycobacteria and will not differentiate between Mycobacterium avium and non-tuberculosis species, between Mycobacterium intracellulare and non-tuberculosis species, or between Mycobacterium tuberculosis-complex bacilli and non-tuberculosis species. For accurate identification of the infecting Mycobacterial species the clinician must rely on culture results which can require anywhere from 3 to 8 weeks of growth followed by extensive biochemical testing. Other tests have been developed based on the detection of metabolic products from Mycobacterium using carbon-14 labelled substrates. In particular, the Bactec (TM) instrument can detect the presence of Mycobacterium within 6 to 10 days of the time of innoculation. Gill, V. J., supra. However, the test does not distinguish Mycobacterium species. It is often important to make this determination so that particular drugs to which the organism is susceptible may be prescribed. For traditional culture methods, this requires an additional 2 to 3 weeks and for the Bactec method, an additional 6 to 10 days.

In addition, specific embodiments for Mycoplasma pneumoniae, the Mycobacterium, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas Group I, bacteria, fungi and Neisseria gonorrhoeae are set forth in the following examples.

As indicated by the below examples, the present invention has significant advantages over each of these prior art methods not only in the enhanced accuracy, specificity and simplicity of the test, but also in greatly reducing the time to achieve a diagnosis. The invention makes possible a definitive diagnosis and initiation of effective treatment on the same day as testing.

EXAMPLE 1

Described below is the preparation of a single strand deoxyoligonucleotide of unique sequence and defined length which is labelled and used as a probe in a solution hybridization assay to detect the presence of rRNA from Mycobacterium avium. This unique sequence is specific for the rRNA of Mycobacterium avium and does not significantly cross-react under the hybridization conditions of this Example, with nucleic acids from any other bacterial species or respiratory infectious agent, including the closely-related Mycobacterium intracellulare. This probe is able to distinguish the two species, notwithstanding an approximate 98% rRNA homology between the two species. In this Example, as well as in Examples 2 and 3, sequences for M. avium, M. tuberculosis complex, M. intracellulare and related organisms were obtained by using a specific primer to a highly conserved region in the 16S rRNA. The sequence of this primer, derived from E. coli rRNA, was 5'-GGC CGT TAC CCC ACC TAC TAG CTA AT-3'. 5 nanograms of primer was mixed with 1 microgram of each rRNA to be sequenced in the presence of 0.1 M KCl and 20. mM Tris-HCl pH 8.3 in a final volume of 10 microliters. The reactions were heated 10 min. at 45° C. and then placed on ice. 2.5 microliters of ³⁵ S dATP and 0.5 microliters of reverse transcriptase were added. The sample was aliquoted into 4 tubes, each tube containing either dideoxy A, G, T, or C. The concentrations of these nucleotides are set forth in Lane et al., supra. The samples were incubated at 40° C. for 30 minutes, and were then precipitated in ethanol, centrifuged and the pellets lyophilized dry. Pellets were resuspended in 10 microliters formamide dyes (100% formamide, 0.1% bromphenol blue and 0.1% xylene cyanol), and loaded onto 80 cm 8% polyacrylamide gels. The gels were run at 2000 volts for 2-4 hours.

Thus, nucleotide sequences for the 16S rRNA of Mycobacterium avium and what were considered to be its closest phylogenetic neighbors, Mycobacterium intracellulare and Mycobacterium tuberculosis, were determined by the method of Lane, D. J. et al., Proc. Nat. Acad. Sci. USA 82:6955 (1985). In addition to determining the rRNA sequences for the organisms noted above, a spectrum of clinically significant Mycobacterium were also sequenced. These included M. fortuitum, M. scrofulaceum and M. chelonae. Selected members of several genera closely related to Mycobacterium were also sequenced, including Rhodococcus bronchialis, Corynebacterium xerosis and Nocardia asteroides.

Partial rRNA sequences from the above organisms were aligned for maximum nucleotide homology, using commercially available software from Intelligenetics, Inc., 1975 El Camino Real West, Mountain View, Calif. 94040-2216 (IFIND Program). From this alignment, regions of sequence unique to Mycobacterium avium were determined. The probe was selected so that it was perfectly complementary to a target nucleic acid sequence and so that it had a 10% or greater mismatch with the aligned rRNA from its known closest phylogenetic neighbor. A sequence 38 bases in length was chosen. The number of mismatched bases relative to the Mycobacterium avium sequence were as follows: Mycobacterium tuberculosis (8); Mycobacterium intracellulare (5); Mycobacterium scrofulaceum (6); Mycobacterium chelonae (12); and Mycobacterium fortuitum (10).

The following cDNA sequence was characterized by the criteria of length, Tm, and sequence analysis as described at pages 7-8 above and was determined to be specific for the rRNA Mycobacterium avium:

ACCGCAAAAGCTTTCCACCAGAAGACATGCGTCTTGAG.

This sequence is complementary to a unique segment found in the 16S rRNA of Mycobacterium avium. The size of the probe is 38 bases. The probe has a Tm of 74° C. and sequence (SEQ ID NO. 5) analysis by the method of Maxam & Gilbert (1980), supra, confirmed that the probe was correctly synthesized. The probe is capable of hybridizing to rRNA of M. avium in the region corresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for Mycobacterium avium, it was tested as a probe in hybridization reactions under the following conditions. ³² P-end-labeled oligonucleotide probes were mixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium avium and reacted in 0.12 M PB hybridization buffer (equimolar amounts of Na₂ HPO₄ and NaH₂ PO₄), 1 mM EDTA and 0.02% SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer both with and without target present. Following separation on hydroxyapatite as outlined in the patent applications identified at page 2, supra, the hybrids were quantitated by scintillation counting. These results are presented in Table 1, showing that the probe has a high extent of reaction to homologous target and very little non-specific binding to the hydroxyapatite.

                  TABLE 1     ______________________________________     HYBRIDIZATION OF THE M. AVIUM PROBE     TO HOMOLOGOUS TARGET rRNA*                   plus rRNA                          minus rRNA     ______________________________________     M. avium probe  85-95%   0.5%     ______________________________________      ##STR1##

Specificity of the probe for M. avium was tested by mixing the ³² P labeled probe with rRNA released from cells of 29 other species of mycobacteria by the sonic disruption techniques described in Murphy et al., U.S. Pat. No. 5,374,522. 1×10⁸ cells were suspended in 0.1 ml 5% SDS and sonicated for 10 minutes at 50-60° C. 1.0 ml of hybridization buffer (45% sodium diisobutyl sulfosuccinate, 40 mM phosphate buffer pH 6.8 and 1 mM EDTA) was added and the mixture incubated for 60 minutes at 72° C. Following incubation, 4.0 ml of hydroxyapatite solution (0.14 M sodium phosphate buffer, pH 6.8, 0.02% SDS and 1.0 gram hydroxyapatite per 50 mls solution) was added and incubated for 5 minutes at 72° C. The sample was centrifuged and the supernatant removed. 4.0 ml wash solution (0.14 M sodium phosphate pH 6.8) was added and sample was vortexed, centrifuged and the supernatant removed. The radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results are shown in Table 2 and indicate that the probe is specific for Mycobacterium avium and does not react with any other mycobacterial species, including Mycobacterium intracellulare.

                  TABLE 2     ______________________________________     HYBRIDIZATION OF THE M. AVIUM PROBE     TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    1.0     M. asiaticum      25276    1.2     M. avium          25291    87.6     M. bovis          19210    1.2     M. bovis (BCG)    19015    1.0     M. chelonae       14472    0.9     M. flavescens     14474    0.9     M. fortuitum       6841    1.0     M. gastri         15754    1.2     M. gordonae       14470    1.2     M. haemophilum    29548    1.3     M. intracallulare 13950    1.5     M. kansasii       12478    1.2     M. malmoense      29571    1.2     M. marinum         827     1.2     M. nonchromogenicum                        1930    1.1     M. phlei          11756    1.3     M. scrofulaceum   19981    1.2     M. shimoidei      27962    2.3     M. simiae         25275    1.2     M. smegmatis      e14468   1.0     M. szulgai        23069    1.0     M. terrae         15755    1.2     M. thermoresistibile                       19527    1.3     M. triviale       23292    1.2     M. tuberculosis (avirulent)                       25177    1.4     M. tuberculosis (virulent)                       27294    1.1     M. ulcerans       19423    1.4     M. vaccae         15483    1.2     M. xenopi         19971    1.5     ______________________________________

As shown in Table 3 the probe also did not react with the rRNA from any of the respiratory pathogens which were also tested by the method just described. Nor did the probe react with any other closely related or phylogenetically more diverse species of bacteria also tested by that method (Table 4).

                  TABLE 3     ______________________________________     HYBRIDIZATION OF M. AVIUM PROBE TO     RESPIRATORY PATHOGENS     Organism          ATCC #   % Probe Bound     ______________________________________     Corynebacterium xerosis                        373     0.7     Fusobacterium nucleatum                       25586    1.3     Haemophilum influenzae                       19418    1.3     Klebsiella pneumoniae                       23357    1.8     Legionella pneunophila                       33152    0.0     Mycoplasma pneumoniae                       15531    3.0     Neisseria meningitidis                       13090    0.0     Pseudomonas aeruginosa                       25330    0.0     Propionibacterium acnes                        6919    1.1     Streptococcus pneumoniae                        6306    0.0     Staphylococcus aureus                       25923    1.5     ______________________________________

                  TABLE 4     ______________________________________     HYBRIDIZATION OF THE M. AVIUM PROBE TO A     PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Acinetobacter calcoaceticus                       33604    0.0     Branhamella catarrahalis                       25238    0.6     Bacillus subtilis 6051     0.9     Bacteroides fragilis                       23745    1.0     Campylobacter jejuni                       33560    0.4     Chromobacterium Violaceum                       29094    1.7     Clostridium perfringens                       13124    2.1     Deinococcus radiodurans                       35073    0.8     Derxia gummosa    15994    0.3     Enterobacter aerogenes                       13048    0.6     Escherichia coli  11775    0.3     Mycobacterium gordonae                       14470    1.9     Mycoplasma hominis                       14027    3.3     Proteus mirabilis 29906    0.0     Psudomonas cepacia                       11762    1.0     Rahnella aquatilis                       33071    2.1     Rhodospirillum rubrum                       11170    0.6     Streptococcus mitis                        9811    0.9     Vibrio parahaemolyticus                       17802    1.2     Yersinia enterocolitica                        9610    0.4     ______________________________________

EXAMPLE 2

After the alignment described in Example 1, the following sequence was characterized by the aforementioned criteria of length, Tm and sequence analysis and was determined to be specific for Mycobacterium intracellulare:

ACCGCAAAAGCTTTCCACCTAAAGACATGCGCCTAAAG

The sequence is complementary to a unique segment found in the 16S rRNA of Mycobacterium intracellulare. The size of the probe was 38 bases. The probe has a Tm of 75° C. and sequence analysis confirmed that the probe was correctly synthesized. The probe hybridizes to RNA of M. intracellulare in the region corresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for the Mycobacterium intracallulare, the probe was tested in hybridization reactions under the following conditions. ³² P-end-labelled oligonucleotide probe was mixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium intracellulare and reacted in 0.12 M PB (equimolar amounts of Na₂ HPO₄ and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer with and without target Mycobacterium intracellulare rRNA present. Following separation on hydroxyapatite as outlined previously the hybrids were quantitated by scintillation counting. These results are shown in Table 5.

                  TABLE 5     ______________________________________     HYBRIDIZATION OF THE M. INTRACELLULARE PROBE     TO HOMOLOGOUS TARGET rRNA*/                     plus rRNA                            minus rRNA     ______________________________________     M. intracellulare probe                       85-95%   0.5%     ______________________________________      ##STR2##

These data shows that the probe has a high extent of reaction to its homologous target and very little non-specific binding to the hydroxyapatite.

Specificity of the Mycobacterium intracellulare probe was tested by mixing the ³² P labelled probe with rRNA released from cells from 29 other species of mycobacteria by sonic disruption techniques described in Murphy et. al. U.S. Pat. No. 5,374,522. All hybridization assays were carried out as described in Example 1. Table 6 indicates that the probe is specific for Mycobacterium intracellulare and does not react with any other mycobacterial species, including Mycobacterium avium. These results are impressive in view of the 98% rRNA homology to M. avium; 98% homology to M kansasii; 98% homology to M. asiaticum; and 97% homology to M. tuberculosis.

                  TABLE 6     ______________________________________     HYBRIDIZATION OF THE M. INTRACELLULARE PROBE     TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    0.9     M. asiaticum      25276    1.1     M. avium          25291    1.3     M. bovis          19210    1.1     M. bovis (BCG)    19015    1.2     M. chelonae       14472    1.0     M. favescens      14474    1.2     M. fortuitum       6841    1.3     M. gastri         15754    1.3     M. gordonae       14470    1.3     M. haemophilum    29548    0.9     M. intracellulare 13950    78.8     M. kansasii       12479    1.1     M. Malmoense      29571    1.0     M. marinum         827     0.9     M. nonchromogenicum                        1930    1.0     M. phlei          11758    1.1     M. scrofulaceum   19981    1.0     M. shimoidei      27962    1.3     M. simiae         25275    1.1     M. smegmatis      e14468   1.3     M. szulgai        23069    1.0     M. terrae         15755    1.4     M. thermoresistibile                       19527    1.6     M. triviale       23292    1.3     M. tuberculosis (avirulent)                       25177    1.2     M. tuberculosis (virulent)                       27294    1.2     M. ulcerans       19423    1.1     M. vaccae         15483    1.0     M. xenopi         19971    1.2     ______________________________________

As shown in Table 7 the probe did not react with the rRNA from any of the respiratory pathogens tested in the hybridization assay. Nor did the probe react with any other closely related or phylogenetically more diverse species of bacteria that were tested (Table 8).

                  TABLE 7     ______________________________________     HYBRIDIZATION OF THE M. INTRACELLULARE PROBE     TO RESPIRATORY PATHOGENS     Organism          ATCC #   % Probe Bound     ______________________________________     Corynebacterium xerosis                        373     2.2     Fusobacterium nucleatum                       25586    1.5     Haemophilum influenzae                       19418    1.3     Klebsiella pneumoniae                       23357    1.2     Legionella pneumophila                       33152    1.2     Mycoplasma pneumoniae                       15531    3.2     Neisseria meningitidis                       13090    1.1     Pseudomonas aeruginosa                       25330    1.0     Propionibacterium acnes                        6919    2.9     Streptococcus pneumoniae                        6306    1.6     Staphylococcus aureus                       25923    1.3     ______________________________________

                  TABLE 8     ______________________________________     HYBRIDIZATION OF THE M. INTRACELLULARE PROBE TO     A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES     Organism           ATTC #   % Probe     ______________________________________     Acinetobacter calcoaceticus                        33604    1.5     Branhamella catarrhalis                        25238    1.8     Bacillus subtilis   6051    1.7     Bacteroides fragilis                        23745    1.9     Campylobacter jejuni                        33560    1.9     Chromobacterium Violaceum                        29094    1.4     Clostridium perfringens                        13124    2.1     Deinococcus radiodurans                        35073    2.1     Derxia gummosa     15994    1.6     Enterobacter aerogenes                        13048    1.3     Escherichia coli   11775    1.2     Mycobacterium gordonae                        14470    2.3     Mycoplasma hominis 14027    2.6     Proteus mirabilis  29906    1.2     Pseudomonas cepacia                        11762    1.7     Rahnella aquatilis 33071    1.5     Rhodospirillum rubrum                        11170    1.4     Strptococcus mitis  9811    1.4     Vibrio parahaemolyticus                        17802    2.5     Yersinia enterocolitica                         9610    1.1     ______________________________________

EXAMPLE 3

After the alignment described in Example 1, the following sequence was characterized by the aforementioned three criteria of size, sequence and Tm, and was determined to be specific to the Mtb complex of organisms, Mycobacterium tuberculosis, Mycobacterium, africanum, Mycobacterium bovis, and Mycobacterium microti:

1. TAAAGCGCTTTCCACCACAAGACATGCATCCCGTG.

The sequence is complementary to a unique segment found in the 16S rRNA of the Mtb-complex bacteria. The size of the probe is 35 bases. The probe has a Tm of 72° C. and sequence analysis confirmed that the probe was correctly synthesized. It is capable of hybridizing in the region corresponding to bases 185-225 of E. coli 16S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex the probe was tested in hybridization reactions under the following conditions. ³² P-end-labelled oligonucleotide probe was mixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium tuberculosis and reacted in 0.12 M PB hybridization buffer (equimolar amounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer with and without target rRNA from Mycobacterium tuberculosis present. Following separation on hydroxyapatite as outlined previously the hybrids were quantitated by scintillation counting. The results are shown in Table 9.

                  TABLE 9     ______________________________________     HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE     TO HOMOLOGOUS TARGET rRNA*/                   plus rRNA                          minus rRNA     ______________________________________     Mtb complex probe                     85-95%   0.5%     ______________________________________      ##STR3##

This data shows that the probe has a high extent of reaction to homologous target and very little non-specific binding to the hydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³² P labelled probe with rRNA released from cells of the 4 Mtb complex bacilli and of 25 other mycobacterial species by sonic disruption techniques described in Murphy et. al., U.S. Pat. No. 5,374,522. All hybridization assays were carried out as described in Example 1. Table 10 indicates that the probe is specific for organisms within the Mtb complex and does not react with any other mycobacterial species.

                  TABLE 10     ______________________________________     HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE     TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    68.1     M. asiaticum      25276    3.4     M. avium          25291    0.9     M. bovis          19210    63.1     M. chelonae       14472    1.1     M. flavescens     14474    0.9     M. fortuitum       6841    1.1     M. gastri         15754    0.8     M. gordonae       14470    1.1     M. haemophilum    29548    0.8     M. intracallulare 13950    1.1     M. kansasii       12479    1.3     M. malmoense      29571    0.9     M. marinum         827     1.1     M. nonchromogenicum                        1930    1.1     M. phlei          11758    1.3     M. scrofulaceum   19981    1.1     M. shimoidei      27962    1.0     M. simiae         25275    1.2     M. smegmatis      e14468   0.9     M. szulgai        23069    1.1     M. terrae         15755    1.0     M. thermoresistibile                       19527    1.0     M. triviale       23292    1.2     M. tuberculosis (avirulent)                       25177    66.2     M. tuberculosis (virulent)                       27294    62.4     M. ulcerans       19423    0.9     M. vaccae         15483    0.8     M. xenopi         19971    2.6     ______________________________________

As shown in Table 11 the probe did not react with the rRNA from any of the respiratory pathogens tested in the hybridization assay. Nor did the probe react with any other closely related or phylogenetically more diverse species of bacteria that were tested (Table 12).

                  TABLE 11     ______________________________________     HYBRIDIZATION OF Mtb-COMPLEX 16S rRNA DNA PROBE     TO RESPIRATORY PATHOGENS     Organism          ATCC #   % Probe Bound     ______________________________________     Corynebacterium xerosis                        373     1.3     Fusobacterium nucleatum                       25586    1.0     Haemophilum influenzae                       19418    1.6     Klebsiella pneumoniae                       23357    1.2     Legionella pneumophila                       33152    1.4     Mycoplasma pneumoniae                       15531    1.1     Neisseria meningitidis                       13090    1.0     Pseudomonas aeruginosa                       25330    1.7     Propionibacterium acnes                        6919    1.2     Streptococcus pneumoniae                       25923    0.9     ______________________________________

                  TABLE 12     ______________________________________     HYBRIDIZATION OF THE Mtb-COMPLEX 16S rRHA DNA     PROBE TO A PHYLOGENETIC CROSS SECTION OF     BACTERIAL SPECIES     Organism           ATCC #   % Probe     ______________________________________     Acinetobacter calcoaceticus                        33604    1.3     Branhamella catarrhalis                        25238    1.5     Bacillus subtilis   6051    1.3     Bacteroides fragilis                        23745    1.3     Campylobacter jejuni                        33560    1.1     Chromobacterium violaceum                        29094    1.0     Clostridium perfringens                        13124    1.2     Deinococcus radiodurans                        35073    1.0     Derxia gummosa     15994    1.0     Enterobacter aerogenes                        13048    1.0     Escherichia coli   11775    1.0     Mycobacterium gordonae                        14470    1.3     Mycoplasma hominis 14027    0.5     Proteus mirabilis  29906    1.0     Pseudomonas cepacia                        11762    2.6     Rahnella aquatilis 33071    1.9     Rhodospirillum rubrum                        11170    1.0     Streptococcus mitis                         9811    1.1     Vibrio parahaemolyticus                        17802    0.9     Yersinia enterocolitica                         9610    1.1     ______________________________________

Two derivatives of the probe of Example 3 (numbered 2-3 below) were made and tested:

2. CCGCTAAAGCGCTTTCCACCACAAGACATGCATCCCG

3. ACACCGCTAAAGCGCTTTCCACCACAAGACATGCATC.

All three probes have similar Tms (72° C.; 73.5° C.; and 72.3° C., respectively) and similar hybridization characteristics.

Hybridization to Mycobacterium tuberculosis complex organisms was 68-75% and non-specific hybridization to hydroxyapatite was less than 2%. Results of hybridization assay tests for these derivatives follow.

                  TABLE 13     ______________________________________     HYBRIDIZATION OF PROBE OF EXAMPLES 3 AND 2     DERIVATIVES THEREOF TO MYCOBACTERIAL SPECIES                     Example                     % Probe 1                            % Probe 2                                     % Probe 3     Organism     ATCC #   Bound    Bound  Bound     ______________________________________     Mycobacterium                  25420    68.1     69.4   70.6      africanum     M. asiaticum 25274    3.4      5.3    1.8     M. avium     25291    0.9      1.6    1.4     M. bovis     19210    63.1     75.3   74     M. chelonae  14472    1.1      1.5    1.6     M. flavescens                  14474    0.9      2.7    1.4     M. fortuitum  6841    1.1      3.6    1.5     M. gastri    15754    0.8      3.6    1.7     M. gordonae  14470    1.1      1.6    1.4     M. haemophilum                  29548    0.8      3.2    1.7     M. intracellulare                  13950    1.1      1.6    1.4     M. kansasii  12478    1.3      2.1    2.0     M. malmoense 29571    0.9      2.8    1.5     M. marinum    827     1.1      2.1    1.5     M. nonchromogenicum                   1930    1.1      3.0    1.5     M. phlei     11758    1.3      1.3    1.1     M. scrofulaceum                  19981    1.1      3.4    1.6     M. shimoidei 27962    1.0      2.7    1.6     M. simiae    25275    1.2      2.9    1.8     M. smegmatis e14468   0.9      1.5    1.2     M. szulgai   23069    1.1      3.6    1.1     M. terrae    15755    1.0      3.7    2.0     M. thermoresistibile                  19527    1.0      1.6    1.3     M. triviale  23292    1.2      1.6    2.0     M. tuberculosis      (avirulent) 25177    66.2     75     68     M. tuberculosis      (virulent)  27294    62.4     74     75     M. ulcerans  19423    0.9      1.7    3.0     M. vaccae    15483    0.8      1.4    1.2     M. xenopi    19971    2.6      1.4    1.2     ______________________________________

EXAMPLE 4

The probe specific for the 23S rRNA of the M. tuberculosis complex was obtained by using a primer which was complementary to a highly conserved region of 23S rRNA. The sequence of this primer, derived from E. coli rRNA, was 5'-AGG AAC CCT TGG GCT TTC GG-3'. Five nanograms of this primer was mixed with 1 microgram of rRNA from M. tuberculosis and other closely related Mycobacterium and the procedure as described for Examples 1, 2 and 3 was followed. After alignment as described in Example 1, the following sequence was determined to be specific to the Mtb complex of organisms, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, and Mycobacterium microti:

TGCCCTACCCACACCCACCACAAGGTGATGT.

The sequence is complementary to a unique segment found in the 23S rRNA of the Mtb-complex bacteria. The oligonucleotide probe was characterized as previously described by the criteria of length, Tm and sequence analysis. The size of the probe is 31 bases. The probe has a Tm of 72.5° C. and sequence analysis confirmed that the probe was correctly synthesized. It is capable of hybridizing in the region corresponding to bases 1155-1190 of E. coli 23S rRNA.

To demonstrate the reactivity of this sequence for the Mtb complex the probe was tested in hybridization reactions under the following conditions. ³² P-end-labelled oligonucleotide probes were mixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycobacterium tuberculosis and reacted in 0.12 M PB hybridization buffer (equimolar amounts of Na₂ HPO₄, and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecy sulfate) at 65° C. for 60 minutes in a final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer with and without target rRNA from Mycobacterium tuberculosis present. Following separation on hydroxyapatite as outlined previously the hybrids were quantitated by scintillation counting. The results are shown in Table 14.

                  TABLE 14     ______________________________________     HYBRIDIZATION OF THE Mtb-COMPLEX     23S rRNA DNA PROBE TO HOMOLOGOUS TARGET rRNA                     plus rRNA                            minus rRNA     ______________________________________     Mtb complex 23S probe                       94%      1.2%     ______________________________________

These data show that the probe has a high extent of reaction to homologous target and very little non-specific binding to the hydroxyapatite.

Specificity of the probe for the Mtb complex was tested by mixing the ³² P labelled probe with rRNA released from cells of the four Mtb complex bacilli and of 25 other mycobacterial species by sonic disruption techniques described in Murphy et al., U.S. Pat. No. 5,374,522. All hybridization assays were carried out as described in Example 1. Table 14 indicates that the probe is specific for organisms within the Mtb complex and does not react with any other mycobacterial species.

                  TABLE 15     ______________________________________     HYBRIDIZATION OF Mtb-COMPLEX 23S rRNA DNA PROBE     TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    33.6     M. asiaticum      25276    1.2     M. avium          25291    1.0     M. bovis          19210    32.0     M. chelonae       14472    1.2     M. flavescens     14474    1.2     M. fortuitum       6841    1.3     M. gastri         15754    1.1     M. gordonae       14470    1.2     M. haemophilum    29548    1.2     M. intracellulare 13950    1.1     M. kansasii       12479    1.3     M. malmoense      29571    1.3     M. marinum         827     1.2     M. nonchromogenicum                        1930    1.0     M. phlei          11758    1.0     M. scrofulaceum   19981    1.1     M. shimoidei      27962    1.2     M. simiae         25275    1.3     M. smegmatis      e14468   1.1     M. szulgai        23069    1.1     M. terrae         15755    1.0     M. thermoresistibile                       19527    1.2     M. triviale       23292    1.0     M. tuberculosis (avirulent)                       25177    33.7     M. tuberculosis (virulent)                       27294    38.1     M. ulcerans       19423    1.3     M. vaccae         15483    1.0     M. xenopi         19971    1.3     ______________________________________

EXAMPLE 5

Three additional Mycobacterium tuberculosis complex probes, Examples 5-7 herein, were identified using two unique primers complementary to 23S rRNA. The first sequence is:

CCATCACCACCCTCCTCCGGAGAGGAAAAGG.

The sequence of this Example 5 was obtained using a 23S primer with the sequence 5'-GGC CAT TAG ATC ACT CC-3'. It was characterized and shown to be specific for the Mycobacterium tuberculosis complex of organisms including Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis. This sequence, from 23S rRNA, is 31 bases in length and has a Tm of 72° C. This probe is capable of hybridizing to RNA of the aforementioned organisms in the region corresponding to bases 540-575 of E. coli 23S rRNA.

To demonstrate the reactivity and specificity of this probe for Mycobacterium tuberculosis complex, it was tested as a probe in hybridization reactions under the following conditions. ³² P-end-labeled oligonucleotide probe was mixed with rRNA released from cells of 30 species of mycobacteria by the sonic disruption techniques described in Murphy et al., U.S. Pat. No. 5,374,522. 3×10⁷ cells were suspended in 0.1 ml 5% SDS and sonicated for 15 minutes at 50-60° C. One ml of hybridization buffer (45% diisobutyl sulfosuccinate, 40 mM phosphate buffer pH 6.8, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubated at 72° C. for 2 hours. Following incubation, 4 ml of 2% (w/v) hydroxyapatite, 0.12 M sodium phosphate buffer pH 6.8, 0.02% SDS, 0.02% sodium azide was added and incubated at 72° C. for 5 minutes. The sample was centrifuged and the supernatant removed. Four ml wash solution (0.12 M sodium phosphate buffer pH 6.8, 0.02% SDS, 0.02% sodium azide) was added and the sample was vortexed, centrifuged and the supernatant removed. The radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results are shown in Table 16 and indicate that the probe is specific for the Mycobacterium tuberculosis complex of organisms.

                  TABLE 16     ______________________________________     HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX     PROBE OF EXAMPLE 5 TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    18.0     M. asiaticum      25274    2.6     M. avium          25291    3.4     M. bovis          19210    21.7     M. bovis (BCG)    35734    35.3     M. chelonae       14472    3.8     M. flavescens     14474    2.3     M. fortuitum       6841    1.8     M. gastri         15754    2.2     M. gordonae       14470    2.8     M. haemophilum    29548    2.8     M. intracellulare 13950    2.1     M. kansasii       12478    1.6     M. malmoense      29571    2.3     M. marinum         827     2.1     M. nonchromogenicum                        1930    2.3     M. phlei          11758    2.1     M. scrofulaceum   19981    2.2     M. shimoidei      27962    1.9     M. simiae         25275    2.2     M. smegmatis      e14468   2.0     M. szulgai        23069    2.2     M. terrae         15755    2.2     M. thermoresistible                       19527    2.2     M. triviale       23292    2.0     M. tuberculosis (avirulent)                       25177    26.4     M. tuberculosis (virulent)                       27294    36.6     M. ulcerans       19423    2.5     M. vaccae         15483    2.4     M. xenopi         19971    2.8     ______________________________________

Table 16 shows that the probe also did not cross react with RNA from any of the closely related organisms tested by the method just described.

                  TABLE 17     ______________________________________     HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX     PROBE OF EXAMPLE 5 TO PHYLOGENETICALLY     CLOSELY RELATED ORGANISMS     Organism          ATCC #   % Probe Bound     ______________________________________     Actinomadura madurae                       19425    2.1     Actinoplanes italicus                       10049    3.1     Arthrobacter oxidans                       14358    2.1     Brevibacterium linens                       e9172    1.9     Corynebacterium xerosis                        373     2.2     Dermatophilus congolensis                       14367    2.2     Microbacterium lacticum                        8180    2.1     Nocardia asteroides                       19247    2.0     Nocardia brasiliensis                       19296    2.2     Nocardia otitidis-caviarum                       14629    2.0     Nocardioposis dassonvillei                       23218    4.0     Oerskovia turbata 33225    2.2     Oerskovia xanthineolytica                       27402    2.0     Rhodococcus aichiensis                       33611    1.9     Rhodococcus aurantiacus                       25938    2.0     Rhodococcus bronchialis                       25592    2.1     Rhodococcus chubuensis                       33609    2.3     Rnodococcus equi   6939    2.4     Rhodococcus obuensis                       33610    2.2     Rhodococcus sputi 29627    2.3     ______________________________________

EXAMPLE 6

The second Mycobacterium tuberculosis complex probe was obtained using a 23S primer with the sequence 5' CCT GAT TGC CGT CCA GGT TGA GGG AAC CTT TGG G-3'. Its sequence is:

CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC

This sequence, from 23S rRNA, is 38 bases in length and has a Tm of 75° C. It hybridizes in the region corresponding to bases 2195-2235 of E. coli 23S rRNA.

Like the complex probe in Example 5, this sequence was characterized and shown to be specific for the Mycobacterium tuberculosis complex of organisms including Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis.

To demonstrate the reactivity and specificity of the probe of this Example 6 to Mycobacterium tuberculosis complex it was tested as a probe in hybridization reactions under the conditions described for the probe in Example 5. The results are shown in Table 18 and indicate that the probe is specific for the Mycobacterium tuberculosis complex of organisms with the exception of Mycobacterium thermoresistibile, a rare isolate which is not a human pathogen.

                  TABLE 18     ______________________________________     HYBRIDIZATION OF THE M. TUBERCULOSIS COMPLEX     PROBE OF EXAMPLE 6 TO MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    56.0     M. asiaticum      25274    3.1     M. avium          25291    2.6     M. bovis          19210    48.0     M. bovis (BCG)    35734    63.0     M. chelonae       14472    2.8     M. flavescens     14474    2.8     M. fortuitum       6841    3.0     M. gastri         15754    3.2     M. gordonae       14470    3.0     M. haemophilum    29548    3.0     M. intracellulare 13950    3.6     M. kansasii       12478    3.9     M. malmoense      29571    2.9     M. marinum         827     2.9     M. nonchromogenicum                        1930    4.8     M. phlei          11758    2.9     M. scrofulaceum   19981    2.6     M. shimoidei      27962    3.6     M. simiae         25275    3.3     M. smegmatis      e14468   3.0     M. szulgai        23069    2.8     M. terrae         15755    2.8     M. thermoresistibile                       19527    11.7     M. triviale       23292    3.2     M. tuberculosis (avirulent)                       25177    65.0     M. tuberculosis (virulent)                       27294    53.0     M. ulcerans       19423    2.5     M. vaccae         15483    2.8     M. xenopi         19971    3.3     ______________________________________

Table 19 shows that the probe also did not cross react with RNA from any of the phylogenetically closely related organisms tested by the method just described.

                  TABLE 19     ______________________________________     HYBRIDIZATION OF THE M. TUBERCULOSIS     COMPLEX PROBE OF EXAMPLE 6 TO PHYLOGENETICALLY     CLOSELY RELATED ORGANISMS     Organism          ATCC #   % Probe Bound     ______________________________________     Actinomadura madurae                       19425    1.3     Actinoplanes italicus                       10049    0.6     Arthrobacter oxidans                       14358    1.1     Brevibacterium linens                       e9172    0.8     Corynebacterium xerosis                        373     1.0     Dermatophilus congolensis                       14367    0.6     Microbacterium lacticum                        8180    1.9     Nocardia asteroides                       19247    0.9     Nocardia brasiliensis                       19296    0.8     Nocardia otitidis-caviarum                       14629    1.5     Nocardioposis dassonvillei                       23218    0.5     Oerskovia turbata 33225    0.3     Oerskovia xanthineolytica                       27402    0.8     Rhodococcus aichiensis                       33611    1.6     Rhodococcus aurantiacus                       25938    0.7     Rhodococcus bronchialis                       25592    1.5     Rhodococcus chubuensis                       33609    0.8     Rhodococcus equi   6939    0.3     Rhodococcus obuensis                       33610    0.8     Rhodococcus sputi 29627    1.4     ______________________________________

EXAMPLE 7

The following additional Mycobacterium tuberculosis complex probe also has been identified using a 23S primer with the same sequence as that of Example 6, namely, 5'-CCT GAT TGC CGT CCA GGT TGA GGG AAC CTT TGG G-3':

AGGCACTGTCCCTAAACCCGATTCAGGGTTC.

This sequence, from 23S rRNA is 31 bases in length and has a Tm of 71° C. It hybridizes in the region corresponding to bases 2195-2235 of E. coli 23S rRNA. As is the case with the Mycobacterium tuberculosis complex probes of Examples 5 and 6 herein, this sequence also was characterized and shown to be specific for the Mycobacterium tuberculosis complex of organisms, including Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis.

To demonstrate the reactivity and specificity of this probe for Mycobacterium tuberculosis complex, it was tested as a probe in hybridization reactions under the conditions described for the probe of Example 5. Table 20 shows that the probe is specific for the Mycobacterium tuberculosis complex of organisms.

                  TABLE 20     ______________________________________     HYBRIDIZATION OF THE MYCOBACTERIUM     TUBERCULOSIS COMPLEX PROBE OF EXAMPLE 7 TO     MYCOBACTERIAL SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Mycobacterium africanum                       25420    43.0     M. asiaticum      25274    0.6     M. avium          25291    0.7     M. bovis          19210    43.0     M. bovis (BCG)    35734    46.0     M. chelonae       14472    0.6     M. flavescens     14474    0.6     M. fortuitum       6841    0.5     M. gastri         15754    0.9     M. gordonae       14470    0.7     M. haemophilum    29548    0.6     M. intracellulare 13950    0.6     M. kansasii       12478    0.9     M. malmoense      29571    0.8     M. marinum         827     0.7     M. nonchromogenicum                        1930    0.8     M. phlei          11758    0.6     M. scrofulaceum   19981    0.7     M. shimoidei      27962    0.8     M. simiae         25275    0.7     M. smegmatis      e14468   0.6     M. szulgai        23069    0.6     M. terrae         15755    0.7     M. thermoresistibile                       19527    0.9     M. triviale       23292    0.7     M. tuberculosis (avirulent)                       25177    40.0     M. tuberculosis (virulent)                       27294    50.0     M. ulcerans       19423    0.7     M. vaccae         15483    0.4     M. xenopi         19971    0.6     ______________________________________

Table 21 shows that the probe also did not cross react with RNA from any of the closely related organisms tested by the method just described.

                  TABLE 21     ______________________________________     HYBRIDIZATION OF THE M. TUBERCULOSIS     COMPLEX PROBE OF EXAMPLE 7 TO PHYLOGENETICALLY     CLOSELY RELATED ORGANISMS     Organism          ATCC #   % Probe Bound     ______________________________________     Actinomadura madurae                       19425    1.0     Actinoplanes italicus                       10049    0.6     Arthrobacter oxidans                       14358    0.4     Brevibacterium linens                       e9172    0.8     Corynebacterium xerosis                        373     0.6     Dermatophilus congolensis                       14367    0.8     Microbacterium lacticum                        8180    0.5     Nocardia asteroides                       19247    0.7     Nocardia brasiliensis                       19296    0.5     Nocardia otitidis-caviarum                       14629    0.6     Nocardioposis dassonvillei                       23218    0.6     Oerskovia turbata 33225    0.8     Oerskovia xanthineolytica                       27402    0.6     Rhodococcus aichiensis                       33611    0.7     Rhodococcus aurantiacus                       25938    0.7     Rhodococcus bronchialis                       25592    0.6     Rhodococcus chubuensis                       33609    0.6     Rhodococcus equi   6939    0.6     Rhodococus obuensis                       33610    0.6     Rhodococcus sputi 29627    0.9     ______________________________________

Notably, overlapping probes may have identical specificity. Compare, for example, the probes of Examples 6 and 7:

Ex. 6 CTGTCCCTAAACCCGATTCAGGGTTCGAGGTTAGATGC

Ex. 7 AGGCACTGTCCCTAAACCCGATTCAGGGTTC

There may be several sequences from a particular region which will yield probes with the desired hybridization characteristics. In other cases, one probe sequence may be significantly better than another probe differing by a single base. In general, the greater the sequence difference (% mismatch) between a target and nontarget organism, the more likely one will be able to alter the probe without affecting its usefulness for a specific application. This phenomenon also was demonstrated by the derivative probes in Example 3.

In Example 7, five bases were added to the 5' end of the probe in Example 6, and 12 bases were removed from the 3' end. The two probes have essentially identical hybridization characteristics.

EXAMPLE 8

The Mycobacterium genus is particularly difficult to distinguish from Nocardia, Corynebacterium and Rhodococcus. These genera have common antigens, precipitins and G & C counts. Despite the fact that these organisms also exhibit 92-94% rRNA homology to the above listed Mycobacterium organisms, we have designed probes which detect all members of the genus Mycobacterium without cross reacting to the related genera.

In addition to the Mycobacterium species probes already disclosed, four probes specific for members of the Mycobacterium genus were identified using one primer complementary to 16S rRNA and one primer complementary to 23S rRNA. Sequence 1 was obtained using a 16S primer with the sequence 5'-TTA CTA GCG ATT CCG ACT TCA-3'. Sequences 2, 3 and 4 were obtained using a 23S primer with the sequence 5'-GTG TCG GTT TTG GGT ACG-3'. Sequence 1 is capable of hybridizing to RNA of the genus Mycobacterium in the region corresponding to bases 1025-1060 of E. coli 16S rRNA. Sequences 2-4 hybridize in regions corresponding to the following bases of E. coli 23S rRNA in our numbering system (See FIG. 2); 1440-1475; 1515-1555; 1570-1610 in our numbering system.

The following sequences were characterized and shown to be specific for the genus Mycobacterium:

1. CCA TGC ACC ACC TGC ACA CAG GCC ACA AGG

2. GGC TTG CCC CAG TAT TAC CAC TGA CTG GTA CGG

3. CAC CGA ATT CGC CTC AAC CGG CTA TGC GTC ACC TC

4. GGG GTA CGG CCC GTG TGT GTG CTC GCT AGA GGC

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73+ C. Sequence 2, from 23S rRNA, is 33 bases in length and has a Tm of 75° C. Sequence 3, from 23S rRNA, is 35 bases in length and has a Tm of 76° C. Sequence 4, from 23S rRNA, is 33 bases in length and has a Tm of 73° C.

To demonstrate the reactivity and specificity of probe 1 for members of the genus Mycobacterium, it was tested as a probe in hybridization reactions under the following conditions. ¹²⁵ I-labeled oligonucleotide probe was mixed with rRNA released from cells of 30 species of mycobacteria by the sonic disruption techniques described in Murphy et al., U.S. Pat. No. 5,374,522. 3×10⁷ cells were suspended in 0.1 ml 5% SDS and sonicated for 15 minutes at 50-60° C. One ml of hybridization buffer (45% diisobutyl sulfosuccinate, 40 mM sodium phosphate pH 16.8, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubated at 72° C. for 2 hours. Following incubation, 2 ml of separation solution (containing 2.5 g/l cationic magnetic microspheres, 0.17 M sodium phosphate buffer pH 6.8, 7.5% Triton X-100 (TM), 0.02% sodium azide) was added and incubated at 72° C. for 5 minutes. The RNA:probe hybrids, bound to the magnetic particles, were collected and the supernatant removed. One ml wash solution (0.12 M sodium phosphate buffer pH 6.8, 14% diisobutyl sulfosuccinate, 5% Triton X-100, 0.02% sodium azide) was added, the particles collected and the supernatant removed. This step was repeated two times. The radioactivity bound to the magnetic particles was determined in a gamma counter. The results are shown in Table 22 and indicate that the probes hybridize to organisms in the genus Mycobacterium and that a combination of probes will detect all members of the genus. Table 23 shows that the probes do not react with other closely related bacteria.

                  TABLE 22     ______________________________________     HYBRIDIZATION OF THE MYCOBACTERIUM     PROBES 1-4 TO MYCOBACTERIAL SPECIES                    %     %       %       %                    Probe Probe   Probe   Probe                    1     2       3       4     Organism    ATCC #   Bound   Bound Bound Bound     ______________________________________     Mycobacterium                 25420    41.5    14.7  17.9  26.7      africanum     M. asiaticum                 25274    31.8    20.2  7.9   0.1     M. avium    25291    11.7    34.7  10.1  1.6     M. bovis    19210    19.4    28.4  44.6  20.9     M. bovis (BCG)                 35734    30.0    35.5  17.8  5.6     M. chelonae 14472     8.6     0.7  6.3   0.2     M. flavescens                 14474    29.8    17.7  2.3   0.9     M. fortuitum                  6841    34.7     2.2  4.8   0.2     M. gastri   15754    27.6    65.1  9.6   22.3     M. gordonae 14470    50.7    55.2  3.1   0.4     M. haemophilum                 29548    40.7    60.7  0.4   12.4     M. intracellulare                 13950    38.8    48.3  0.9   5.4     M. kansasii 12478    53.4    27.3  24.5  27.8     M. malmoense                 29571     3.1    38.4  0.8   1.5     M. marinum   827     41.7     4.1  4.8   0.1     M. non-      1930    35.0    42.9  0.5   16.4      chromogenicum     M. phlei    11758    23.7     0.6  1.8   0.6     M. scrofulaceum                 19981    35.1    66.9  0.9   26.4     M. shimoidei                 27962    34.6     1.4  1.3   4.8     M. simiae   25275    45.9    44.0  5.3   0.1     M. smegmatis                 e14468   31.3     4.0  5.6   0.1     M. szulgai  23069    19.4    22.3  1.5   3.0     M. terrae   15755    25.6    21.7  0.4   12.3     M. thermo-  19527    20.3    34.5  3.1   17.6      resistibile     M. triviale 23292    37.3     4.6  4.3   0.1     M. tuberculosis                 25177    38.5    26.3  11.3  23.0      (avirulent)     M. tuberculosis                 27294    13.8    12.4  38.4  22.3      (virulent)     M. ulcerans 19423    33.9    28.7  0.4     M. vaccae   15483     8.8    36.2  4.8   3.2     M. xenopi   19971    38.4     2.1  3.8   0.2     ______________________________________

                  TABLE 23     ______________________________________     HYBRIDIZATION OF THE MYCOBACTERIUM PROBES     1-4 TO PHYLOGENETICALLY CLOSELY     RELATED ORGANISMS                    %     %       %       %                    Probe Probe   Probe   Probe                    1     2       3       4     Organism    ATCC #   Bound   Bound Bound Bound     ______________________________________     Actinomadura                 19425    0.2     0.3   0.2   0.1      madurae     Actinoplanes                 10049    0.4     0.5   0.3   0.2      italicus     Arthrobacter                 14358    0.2     0.4   0.3   0.1      oxidans     Brevibacterium                 e9172    0.3     0.3   0.3   0.1      linens     Corynebacterium                  373     0.4     0.3   0.3   0.1      xerosis     Dermatophilus                 14367    0.4     0.6   0.3   0.2      congolensis     Microbacterium                  8180    0.2     0.3   0.2   0.1      lacticum     Nocardia    19247    0.3     0.3   0.4   0.1      asteroides     Nocardia    19296    0.4     0.3   0.6   0.1      brasiliensis     Nocardia    14629    0.4     0.4   1.0   0.3      otitidis-      caviarum     Nocardioposis                 23218    0.3     0.2   0.3   0.1      dassonvillei     Oerskovia   33225    0.2     0.2   0.3   0.1      turbata     Oerskovia   27402    0.2     0.3   0.3   0.1      xanthineolytica     Rhodococcus 33611    0.4     0.2   0.3   0.2      aichiensis     Rhodococcus 25938    0.3     0.4   0.3   0.2      aurantiacus     Rhodococcus 25592    0.4     0.3   0.3   0.1      bronchialis     Rhodococcus 33609    0.6     0.4   0.3   0.3      chubuensis     Rhodococcus equi                  6939    0.4     0.4   0.4   0.5     Rhodococcus 33610    0.5     0.5   0.3   0.1      obuensis     Rhodococcus sputi                 29627    0.4     0.5   0.4   0.3     ______________________________________

EXAMPLE 9

Mycoplasmas are small, aerobic bacteria lacking cell walls. Mycoplasma pneumoniae is estimated to cause 8-15 million infections per year. The infections may be asymptomatic or range in severity from mild to severe bronchitis and pneumonia. The organism is believed to cause about 10% of pneumonias in the general population and 10-50% of the pneumonias of members of groups in prolonged, close contact such as college students and military personnel.

Diagnosis until now has required isolation of the organism in culture or demonstration of an increase in antibody titer. Culturing of the organism involves inoculation of respiratory tract specimens onto agar or biphasic media containing bacterial growth inhibitors. Examination for growth at 3-4 and 7-10 days is used to establish the presence or absence of any mycoplasma. Mycoplasma pneumoniae must then be identified by hemadsorption (the ability of M. pneumoniae to adhere sheep or guinea pig erythrocytes), hemolysis (the ability of M. pneumoniae to produce beta hemolysis of sheep or guinea pig erythrocytes in blood agar), growth inhibition by specific antibodies, or immunofluorescence with specific antibodies. The present invention has significant advantages over each of these prior art methods both because of the simplicity of the test and because of the greatly reduced time necessary to achieve a diagnosis.

A probe specific for the 5S rRNA of M. pneumoniae was obtained by a comparison of known rRNA sequences. The particular sequences aligned were from M. pneumoniae, M. gallisepticum and Ureaplasma urealyticum (Rogers, M. J. et al. 1985, Proc. Natl. Acad. Sci. USA, 82 (1160-1164), M. capricolum (Hori, H. et al. 1981, Nucl. Acids Res. 9, 5407-5410) and Spiroplasma sp. (Walker, R. T. et al. 1982 Nucl. Acids Res. 10, 6363-6367). The alignments were performed as described above and outlined at page 6. 5S rRNA can be isolated and sequenced as outlined in Rogers et al., or a primer can be made which is complementary to a conserved region in the 5S rRNA and sequencing performed as outlined in Examples 1-4. The conserved region of 5S rRNA is documented in Fox, G. E. and Woese, C. R., 1975, Nature 256: 505-507. The following sequence was determined to be specific for Mycoplasma pneumoniae:

GCTTGGTGCTTTCCTATTCTCACTGAAACAGCTACATTCGGC.

The sequence is complementary to a unique segment found in the 5S rRNA of Mycoplasma pneumoniae in the region corresponding to bases 65-108 of E. coli 5S rRNA, and was selected by comparison to 5S rRNA sequences from Mycoplasma gallisepticum, Spiroplasma mirum and Ureaplasma urealyticum. The oligonucleotide probe was characterized as described above. The size of the probe was 42 bases. The probe has a Tm of 71.5° C.

To demonstrate the reactivity of this sequence for Mycoplasma pneumoniae, the probe was tested in hybridization reactions under the following conditions. ³² P-end-labelled oligonucleotide probe was mixed with 1 microgram (7×10⁻¹³ moles) of purified rRNA from Mycoplasma pneumoniae and reacted in 0.12 M PB (equimolar amounts of Na₂ HPO₄ and NaH₂ PO₄), 1 mM EDTA and 0.2% SDS (sodium dodecyl sulfate) at 65° C. for 60 minutes in a final volume of 50 microliters. In separate tubes the probe was mixed with the hybridization buffer with and without target Mycoplasma pneumoniae rRNA present. Following separation on hydroxyapatite as outlined previously the hybrids were quantitated by scintillation counting. These results are shown in Table 24.

                  TABLE 24     ______________________________________     HYBRIDIZATION OF THE M. PNEUMONIAE 5S rRNA DNA     PROBE TO HOMOLOGOUS TARGET rRNA*/                     plus rRNA                            minus rRNA     ______________________________________     M. pneumoniae 5S probe                       85-95%   0.5%     ______________________________________      ##STR4##     1   This data shows that the probe has a high extent of reaction to its      homologous target and very little non-specific binding to the      hydroxyapatite.

Specificity of the M. pneumoniae 5S probe was tested by mixing the ³² P labelled probe with rRNA released from cells from other Mycoplasma species. All hybridization assays were carried out as described in Example 1. Table 25 indicates that the probe is specific for Mycoplasma pneumoniae and does not react with any other Mycoplasma species.

                  TABLE 25     ______________________________________     HYBRIDIZATION OF M. PNEUMONIAE PROBE TO     OTHER MYCOPLASMA SPECIES     ______________________________________     Acholeplasma laidlawii                        14089   3.3     M. buccale         23636   1.7     M. capricolum      23205   2.4     M. columbinsale    33549   1.4     M. faucium         25293   1.4     M. fermentans      15474   1.0     M. gallisepticum   19610   1.8     M. gallopavonis    33551   1.6     M. genitalium      3353c   1.7     M. hominis         14027   1.3     M. orale           23714   1.8     M. pneumoniae      15531   78.0     M. primatum        15497   1.6     M. salivarium      23064   0.6     Spiroplasma mirum          2.3     ______________________________________

As shown in Table 26, the probe did not react with any other closely related or phylogenetically diverse species of bacteria.

                  TABLE 26     ______________________________________     HYBRIDIZATION OF M. PNEUMONIAE PROBE TO     A PHYLOGENETIC CROSS SECTION OF BACTERIA     Organism           ATCC #     % Probe Bound     ______________________________________     Corynebacterium xerosis                         373       1.4     Haemophilus influenzae                        19418      1.4     Klebsiella pneumoniae                        23357      1.3     Legionella pneumophila                        33152      1.8     Mycobacterium tuberculosis (avir)                        25177      1.6     Mycoplasms pneumoniae                        15531      52     Neisseria meningitidis                        13077      0.6     Propionibacterium acnes                         6919      2.0     Pseudomonas aeruginosa                        25330      1.6     Staphylococcus aureus                        12598      2.0     Streptococcus pneumoniae                        c6306      1.9     ______________________________________

Four additional probe sequences (numbered 2-5 below) specific for Mycoplasma pneumoniae were obtained by utilizing four unique primers complementary to conserved regions on 16S rRNA. The regions correspond, respectively, to bases 190-230; 450-490; 820-860; and 1255-1290 of E. coli 16S rRNA. Probe sequence #1 was obtained using a primer with the sequence 5'-GGCCGTTACCCCACCTACTAGCTAAT-3'. Probe sequence #2 was obtained with a primer with the sequence 5'-GTATTACCGCGGCTGCTGGC-3'. Probe sequence #3 was obtained with a primer with the sequence 5'-CCGCTTGTGCGGGCCCCCGTCAATTC-3'. Probe sequence #4 was obtained using a primer with the sequence 5'-CGATTACTAGCGATTCC-3'. Sequencing reactions were performed as outlined in previous examples. The M. pneumoniae sequences were compared with sequences from Mycoplasma genitalium, Mycoplasma capricolum, Mycoplasma gallisepticum and Spiroplasma mirum.

The following probe sequences were characterized by criteria described in Example 1 of the parent application and were shown to be specific for Mycoplasma Pneumoniae:

2. AATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT

3. CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATT

4. TACCGAGGGGATCGCCCCGACAGCTAGTAT

5. CTTTACAGATTTGCTCACTTTTACAAGCTGGCGAC.

Probe #2 is 35 bases in length and has a Tm of 67° C. Probe #3 is 35 bases in length and has a Tm of 66° C. Probe #4 is 30 bases in length and has a Tm of 69° C. Probe #5 is 35 bases long with a Tm of 66° C.

When the four probes were mixed and used in hybridization assays at 60° C. in the same manner as previous examples, they were found to be specific for M. pneumoniae. The probes do not cross react with other respiratory pathogens or with any organism representing the bacterial phylogenetic tree (Table 28).

                  TABLE 27     ______________________________________     HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE     PROBES 2-5 TO MYCOPLASMA SPECIES     Organism          ATCC #   % Probe Bound     ______________________________________     Acholeplasma axanthum                       27378    0.34     Acholeplasma laidlawii                       14089    0.30     Mycoplasma arginini                       23838    0.20     Mycoplasma arthritidis                       19611    0.49     Mycoplasma bovigenitalium                       19852    0.18     Mycoplasma bovis  25523    0.43     Mycoplasma buccale                       23636    0.37     Mycoplasma californicum                       33451    0.79     Mycoplasma capricolum                       23205    0.38     Mycoplasma columbinasale                       33549    0.54     Mycoplasma columborale                       29258    0.50     Mycoplasma faucium                       25293    0.45     Mycoplasma fermentans                       15474    0.27     Mycoplasma gallisepticum                       19610    0.25     Mycoplasma gallopavonis                       33551    0.47     Mycoplasma genitalium                       33530    2.5     Mycoplasma hominis                       14027    0.52     Mycoplasma hyorhinis                       17981    0.46     Mycoplasma orale  23714    0.56     Mycoplasma pneumoniae                       15531    34.0     Mycoplasma primatum                       15497    0.71     Mycoplasma pulmonis                       19612    0.68     Mycoplasma salivarium                       23064    0.46     Spiroplasma citri 29416    0.60     Spiroplasma mirum 29335    0.52     ______________________________________

                  TABLE 28     ______________________________________     HYBRIDIZATION OF MYCOPLASMA PNEUMONIAE     PROBES 2-5 WITH OTHER BACTERIA     Organism          ATCC #   % Probe Bound     ______________________________________     Actinomyces israelii                       10049    1.0     Bacteroides fragilis                       23745    1.4     Bifidobacterium breve                       15700    1.0     Bordetella bronchiseptica                       10580    0.9     Clostridium innocuum                       14501    1.0     Clostridium pasteurianum                        6013    0.9     Clostridium perfringens                       13124    1.1     Clostridium ramosum                       25582    1.0     Corynebacterium xerosis                        373     0.8     Erysipelothrix rhusiopathiae                       19414    1.1     Escherichia coli  11775    1.0     Haemophilus influenzae                       19418    0.9     Klebsiella pneumoniae                       15531    1.0     Lactobacillus acidophilus                        4356    1.4     Legionella pneumophila                       33154    0.8     Listeria monocytogenes                       15313    1.2     Moraxella osloensis                       19976    1.1     Mycobacterium tuberculosis                       25177    1.0     Neisseria meningitidis                       13077    1.0     Pasteurella multocida                        6529    1.6     Peptococcus magnus                       14955    0.9     Propionibacterium acnes                        6919    1.1     Pseudomonas aeruginosa                       25330    1.0     Staphylococcus aureus                       12600    1.0     Streptococcus faecalis                       19433    1.5     Streptococcus mitis                        9811    1.0     Streptococcus pneumoniae                        6306    1.0     Streptococcus pyogenes                       19615    1.1     ______________________________________

EXAMPLE 10

The genus Legionella contains 22 species which are all potentially pathogenic for humans. These organisms cause Legionnaires' disease, an acute pneumonia, or Pontiac fever, an acute, non-pneumonic, febrile illness that is not fatal.

Legionella species have also been shown to be responsible for nosocomial pneumonia occurring predominantly among immunocompromised patients.

Legionellosis, which includes Legionnaires' disease and Pontiac fever, is diagnosed on the basis of clinical symptoms, either direct or indirect fluorescence antibody tests, and by culture using a buffered charcoal yeast extract (BCYE) agar containing selective antimicrobial agents. There is no single definitive genus test known in the prior art. (See Bergey's Manual of Systematic Bacteriology at page 283, (ed. 1984)). The fluorescent antibody tests are not able to identify all species of Legionella, but only those few for which antibodies exist. The culture method is not definitively diagnostic for Legionella species.

The oligonucleotide sequences described below, when used as probes in a nucleic acid hybridization assay, accurately identify all species of Legionella. This assay is more sensitive than culture or antibody tests and shortens significantly the time of identification and, thus, diagnosis. The assay, therefore, represents a significant improvement over prior diagnostic methods.

Three probe sequences specific for the genus Legionella were obtained by utilizing three unique primers complementary to conserved regions on both 16S and 23S rRNA. Sequence 1 was obtained by using a 16S primer with the sequence 5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2 was obtained with a 23S primer of sequence 5'-CAG TCA GGA GTA TTT AGC CTT-3'. Probe sequence 3 was obtained with a 16S primer of sequence 5'GCT CGT TGC GGG ACT TAA CCC ACC AT-3'. Sequencing with these primers was performed as described for previous examples.

The following three sequences were characterized by the criteria described in Example 1 and were shown to be specific for the genus Legionella. The phylogenetically nearest neighbors Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus and Acinetobacter calcoaceticus were used as comparisons with sequences from Legionella species.

1. TACCCTCTCCCATACTCGAGTCAACCAGTATTATCTGACC

2. GGATTTCACGTGTCCCGGCCTACTTGTTCGGGTGCGTAGTTC

3. CATCTCTGCAAAATTCACTGTATGTCAAGGGTAGGTAAGG.

Sequence 1, from 16S rRNA, is 40 bases in length and has a Tm of 72° C. Sequence 2, from 23S rRNA, is 42 bases in length and has a Tm of 73° C. Sequence 3, from 16S rRNA, is 40 bases in length and has a Tm of 68° C. These sequences are capable of hybridizing to RNA of the genus Legionella in the regions corresponding respectively to, 630-675 of E. coli 16S rRNA; 350-395 of E. coli 23S rRNA; and 975-1020 of E. coli 16S rRNA. When mixed together the probes had a combined average Tm of 73° C. Analysis on polyacrylamide gels showed that each probe was the correct length and sequence analysis demonstrated that each was the correct sequence of bases.

When the three probes were mixed and used in a hybridization assay, they were found to be specific for the genus Legionella (Tables 29 and 30) and did not cross react with other respiratory pathogens or with any selected organism from the phylogenetic tree (Tables 31 and 32). Use of more than one probe, i.e., a mixture of probes, can result in increased assay sensitivity and/or in an increase in the number of non-viral organisms to be detected.

                  TABLE 29     ______________________________________     HYBRIDIZATION OF LEGIONELLA     PROBES TO HOMOLOGOUS TARGET rRNA                   plus rRNA                          minus rRNA     ______________________________________     Legionella probe                     80%      1.0%     ______________________________________

                  TABLE 30     ______________________________________     HYBRIDIZATION OF LEGIONELLA     PROBES TO LEGIONELLA SPECIES     Organism        ATCC #      % Probes Bound     ______________________________________     L. anisa        35292       42.0     L. bozemanii    33217       58.0     L. cherrii      35252       69.0     L. dumoffii     33279       57.0     L. erythra      CDC#9PlW044C                                 26.0     L. feeleii      35303       59.0     L. hackeliae    35250       47.0     L. jamestowniensis                     35298       20.0     L. jordanis     33623       50.6     L. longbeachae  33484       48.0     L. maceachernii 35300       25.0     L. micdadei     33704       38.0     L. oakridgensis 33761       44.0     L. parisiensis   9060       69.0     L. pneumophila 1*                      6736       75.0     L. pneumophila 2            64.0     L. pneumophila 3            73.0     L. pneumophila 4            73.0     L. pneumophila 5            78.0     L. pneumophila 6            75.0     L. pneumophila 7            73.0     L. pneumophila 8            63.0     L. pneumophila 11           75.0     L. rubrilucens  35304       12.0     L. sainthelensi 35248       61.0     L. sainticrucis 35301       24.0     L. spiritensis  CDC#MSH9    55.0     L. steigerwaltii                      7430       56.0     L. wadsworthii  33877       37.0     ______________________________________      *The numbers 1-8 and 11 are serotypes of L. pneumophila.-

                  TABLE 31     ______________________________________     HYBRIDIZATION OF LEGIONELLA PROBES TO     RESPIRATORY PATHOGENS     Organisms         ATCC #   % Probe Bound     ______________________________________     Corynebacterium xerosis                        373     2.1     Haemophilus influenzae                       19418    2.3     Klebsiella pneumoniae                       23357    2.0     Mycoplasma pneumoniae                       15531    2.3     Neisseria meningitidis                       13090    2.2     Pseudomonas aeruginosa                       25330    1.2     Propionibacterium acnes                        6919    1.6     Streptococcus pneumoniae                        6306    0.8     Staphylococcus aureus                       25923    1.6     ______________________________________

                  TABLE 32     ______________________________________     HYBRIDIZATION OF LEGIONELLA PROBES TO     A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES     Organisms         ATCC #   % Probe Bound     ______________________________________     Acinetobacter calcoaceticus                       33604    1.4     Branhamella catarrahalis                       25238    2.0     Bacillus subtilis  6051    1.9     Bacteroides fragilis                       23745    2.2     Campylobacter jejuni                       33560    1.2     Chromobacterium violaceum                       29094    1.3     Clostridium perfringens                       13124    1.9     Deinoccoccus radiodurans                       35073    1.8     Derxia gummosa    15994    2.0     Enterobacter aerogenes                       13048    1.4     Escherichia coli  11775    1.2     Mycoplasma hominis                       14027    1.1     Proteus mirabilis 29906    1.4     Pseudomonas cepacia                       11762    1.1     Rahnella aquatilis                       33071    1.7     Rhodospirillum rubrum                       11170    2.0     Streptococcus mitis                        9811    2.0     Vibrio parahaemolyticus                       17802    2.0     Yersinia enterocolitica                        9610    1.2     ______________________________________

Three additional probe sequences (numbered 4-6) specific for the genus Legionella were obtained by utilizing two primers complementary to conserved regions on 23S rRNA. Sequence 4 was made from a 23S primer with the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Probe sequences 5 and 6 were made from a 23S primer of sequence 5'-AAG CCG GTT ATC CCC GGG GTA ACT TTT-3". Sequencing with these primers was performed as described for previous examples.

The following three sequences were characterized by the criteria previously described and were shown to be specific for the genus Legionella. The phylogenetically nearest neighbors Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus and Actinetobacter calcoaceticus were used for comparisons with sequences from Lecionella species.

4. GCG GTA CGG TTC TCT ATA AGT TAT GGC TAG C

5. GTA CCG AGG GTA CCT TTG TGC T

6. CAC TCT TGG TAC GAT GTC CGA C

Probe 4, complementary to 23S rRNA in the region corresponding to bases 1585-1620 of E. coli 23S rRNA, is 31 bases long and has a Tm of 67° C. Probe 5, complementary to 23S rRNA in the region corresponding to bases 2280-2330 of E. coli 23S rRNA, is 22 bases long and has a Tm of 66° C. Probe 6, complementary to 23S rRNA in the same region as Probe 5, is 22 bases long and has a Tm of 63° C.

When the three probes were mixed with probe 3 above and used in a hybridization assay as described for probes 1-3, they were found to be specific for the genus Legionella (Table 33) and did not cross react with other respiratory pathogens or with any selected organism from the phylogenetic tree (Tables 34 and 35). Using more than one probe, i.e., a mixture of probes, can improve assay sensitivity and/or increase the number of non-viral organisms detected.

                  TABLE 33     ______________________________________     HYBRIDIZATION OF LEGIONELLA PROBES TO     LEGIONELLA SPECIES     Organism     ATCC#         % Probes Bound     ______________________________________     L. anisa     35292         29.6     L. bozemanii 33217         35.5     L. cherrii   35252         29.2     L. dumoffii  33279         26.0     L. erythra   35303         32.0     L. feelii    CDC#9P1WO44C  32.0     L. hackeliae 35250         39.0     L. jamestowniensis                  35298         31.2     L. jordanis  33623         25.7     L. longbeachae                  33484         27.6     L. maceahernii                  35300         39.3     L. micdadei  33204         31.0     L. oakridgensis                  33761         24.4     L. parisiensi                  35299         31.2     L. pneumophila 1*                  33153         40.0     L. pneumophila 2                  33154         38.5     L. pneumophila 3                  33155         44.6     L. pneumophila 4                  33156         48.6     L. pneumophila 5                  33216         32.0     L. pneumophila 6                  33215         43.0     L. pneumophila 7                  33823         29.5     L. pneumophila 8                  35096         37.6     L. pneumophila 11                  43130         44.5     L. rubrilucens                  35304         30.1     L. sainthelensis                  35248         27.0     L. sainticrucis                  35301         22.0     L. spiritensis                  CDC#MSH9      40.5     L. steigerwaltii                  35302         31.7     L. wadsworthii                  33877         30.0     ______________________________________      * The numbers 1-8 and 11 are serotypes of L. pneumophila.

                  TABLE 34     ______________________________________     HYBRIDIZATION OF LEGIONELLA PROBES TO     RESPIRATORY PATHOGENS     Organisms        ATCC#   % Probe Bound     ______________________________________     Corynebacterium xerosis                       373    0.13     Haemophilum influenzae                      19418   0.12     Klebsiella pneumoniae                      23357   0.13     Neisseria meningitidis                      13090   0.14     Pseudomonas aeruginosa                      25330   0.13     Propionibacterium acnes                       6919   0.11     Streptococcus pneumoniae                       6306   0.08     Staphylococcus aureus                      25923   0.15     ______________________________________

                  TABLE 35     ______________________________________     HYBRIDIZATION OF LEGIONELLA PROBES TO     A PHYLOGENETIC CROSS SECTION OF BACTERIAL SPECIES     Organisms         ATCC#   % Probe Bound     ______________________________________     Acinetobacter calcoaceticus                       33604   0.12     Branhamella catarrahalis                       25238   0.13     Bacillus subtilis  6051   0.09     Bacteroides fragilis                       23745   0.12     Campylobacter jejuni                       33560   0.06     Chromobacterium violaceum                       29094   0.33     Clostridium perfringens                       13124   0.07     Deinoccoccus radiodurans                       35073   0.11     Derxia gummosa    15994   0.15     Enterobacter aerogenes                       13048   0.26     Escherichia coli  11775   0.09     Mycoplasma hominis                       14027   0.09     Proteus mirabilis 29906   0.09     Pseudomonas cepacia                       17762   0.20     Rahnella aquatilis                       33071   0.15     Rhodospirillum rubrum                       11170   0.13     Streptococcus mitis                        9811   0.07     Vibrio parahaemolyticus                       17802   0.11     Yersinia enterocolitica                        9610   0.19     ______________________________________

EXAMPLE 11

Chlamydia are gram-negative, non-motile, obligate intracellular bacteria. The species C. trachomatis is associated with endemic trachoma (the most common preventable form of blindness), inclusion conjunctivitis and lymphogranuloma venereum (LGV). It is a major cause of nongonococcal urethritis in men and may cause cervicitis and acute salpingitis in women. Eye disease or chlamydial pneumonia may develop in newborns passing through the infected birth canal.

There are several methods known in the art for identification of C. trachomatis in the urogenital tract, for example, by direct immunofluorescent staining or enzyme immunoassay of clinical specimens. The method of choice, however, remains culture of the organism in cycloheximide treated McCoy cells. Cell culture is followed by morphological or fluorescent antibody staining for confirmation of the organism's identity.

The inventive oligonucleotide sequences described below, when used as probes in nucleic acid hybridization assay, accurately identify Chlamydia trachomatis isolates. This assay test is equal in sensitivity to culture or antibody tests and, in the case of culture, significantly shortens the time to identification, and thus, diagnosis.

The use of probes to identify and distinguish between members of the species is novel and inventive. Indeed, Kingsbury, D. T., and E. Weiss, 1968 J. Bacteriol. 96: 1421-23 (1968); Moulder, J. W., ASM News, Vol.50, No.8, (1984) report a 10% DNA homology between C. trachomatis and C. psittaci. Moreover, these reports show that different C. trachomatis strains differ in DNA homology. Weisberg, W. G. et. al, J. Bacteriol. 167:570-574 (1986) published the 16S rRNA sequences of C. psittaci and noted that C. trachomatis and C. psittaci share a greater than 95% rRNA homology. From these reports, it may be inferred that it would be difficult to invent (1) probes capable of hybridizing to all strains of C. trachomatis; and (2) probes capable of distinguishing between C. trachomatis and C. psittaci. The following probes accomplish both objectives.

Ten probe sequences specific for Chlamydia trachomatis were made using seven unique primers complementary to conserved regions of both 16S and 23S rRNA. Probe sequence 1 was obtained from a 16S primer of sequence 5'-TCT ACG CAT TTC ACC GCT ACA C-3'. Probe sequence 2 was obtained with a 16S primer of sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. Sequences 3 and 4 were obtained using a 16S primer with the sequence 5'-GGC CGT TAC CCC ACC TAC TAG CTA AT-3'. Probe sequences 5 and 6 were obtained with a 23S primer of sequence 5'-CTT TCC CTC ACG GTA-3'. Probe sequences 7 and 8 were obtained with a 23S primer of sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Probe sequence 9 was obtained with a 23S primer of sequence 5'-TCG GAA CTT ACC CGA CAA GGA ATT TC-3'. Probe sequence 10 was obtained with a primer of sequence 5'-CTA CTT TCC TGC GTC A-3'.

The following ten sequences were characterized using the criteria described in Example 1 and were shown to be specific for the rRNA of Chlamydia trachomatis. The phylogenetically nearest neighbor Chlamydia psittaci was used for comparison with Chlamydia trachomatis sequence.

1. CCG ACT CGG GGT TGA GCC CAT CTT TGA CAA

2. TTA CGT CCG ACA CGG ATG GGG TTG AGA CCA TC

3. CCG CCA CTA AAC AAT CGT CGA AAC AAT TGC TCC GTT CGA

4. CGT TAC TCG GAT GCC CAA ATA TCG CCA CAT TCG

5. CAT CCA TCT TTC CAG ATG TGT TCA ACT AGG AGT CCT GAT CC

6. GAG GTC GGT CTT TCT CTC CTT TCG TCT ACG

7. CCG TTC TCA TCG CTC TAC GGA CTC TTC CAA TCG

8. CGA AGA TTC CCC TTG ATC GCG ACC TGA TCT

9. CCG GGG CTC CTA TCG TTC CAT AGT CAC CCT AAA AG

10. TAC CGC GTG TCT TAT CGA CAC ACC CGC G

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 66° C. Sequence 2, from 16S rRNA, is 32 bases in length and has a Tm of 67° C. Sequence 3, from 16S rRNA, is 39 bases in length and has a Tm of 70° C. Sequence 4, from 16S rRNA, is 33 bases in length and has a Tm of 69° C. Sequence 5, from 23S rRNA, is 41 bases in length and has a Tm of 71° C. Sequence 6, from 23S rRNA, is 30 bases in length and has a Tm of 72° C. Sequence 7, from 23S rRNA, is 33 bases in length and has a Tm of 72° C. Sequence 8, from 23S rRNA, is 30 bases in length and has a Tm of 71° C. Sequence 9, from 23S rRNA is 35 bases in length and has a Tm of 74° C. Sequence 10 is 28 bases in length and has a Tm of 72° C.

The reactivity and specificity of the probes was tested hybridization assays. ³² P-end-labeled oligonucleotide probes 1 and 2 were mixed with purified RNA or RNA released from at least 10⁷ organisms in 0.55 ml of 41% diisobutyl sulfosuccinate, 3% sodium dodecyl sulfate, 0.03 M sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA at 60° C. (probe 1) or 64° C. (probe 2) for 1 hour. Hybrids were bound to hydroxyapatite as described in previous examples and the amount of radioactivity bound was determined by scintillation counting. Table 36 shows that probes 1 and 2 hybridize well to all serotypes of C. trachomatis tested. Probe 1 does not react with any strain of C. psittaci tested and probe 2 does not react with two of the strains. Probe 2 does react with the ovine polyarthritis strain of C. psittaci, an organism which is not known to infect humans. Table 37 demonstrates the reactivity and specificity of probes 3-9 when ¹²⁵ I-labeled and used as a mix. In this case, the hybrids were bound to cationic magnetic particles as described in Arnold et al., U.S. patent application Ser. No. 020,866 filed Mar. 2, 1987. These probes hybridize well to all strains of C. trachomatis tested and not to any strains of C. psittaci. Probes 3-9 were further tested against a panel of organisms commonly found in the urogenital tract (Table 38) and a phylogenetic cross section of organisms (Table 39). In all cases, the probes were shown to be specific. Probe 10 is 25% non-homologous to C. psittaci and also should be specific for C. trachomatis.

                  TABLE 36     ______________________________________     HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS     PROBES 1 AND 2 TO CHLAMYDIA RNA                           % Probe Bound     Organism          ATCC#     Probe 1  Probe 2     ______________________________________     Chlamydia trachomatis serotype C                       VR578     22       39     Chlamydia trachomatis serotype E                       VR348B    27       48     Chlamydia trachomatis serotype G                       VR878     20       44     Chlamydia trachomatis serotype I                       VR880     20       42     Chlamydia trachomatis serotype K                       VR887     28       45     Chlamydia psittaci guinea pig                       VR813     1.2      1.4     conjunctivitis strain     Chlamydia psittaci ovine                       VR656     1.0      3.0     abortion strain     Chlamydia psittaci ovine poly-                       VR619     1.1      35.3     arthritis strain     ______________________________________

                  TABLE 37     ______________________________________     HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9     WITH CHLAMYDIA rRNA                                    Ratio Counts     Organism   Serovar    ATCC #   Bound*     ______________________________________     C. trachomatis                A                   689     C. trachomatis                B                   560     C. trachomatis                Ba                  1066     C. trachomatis                C          VR548    962     C. trachomatis                D                   1192     C. trachomatis                E          VR348    1022     C. trachomatis                F                   391     C. trachomatis                G          VR878    874     C. trachomatis                H                   954     C. trachomatis                I          VR880    943     C. trachomatis                J                   482     C. trachomatis                K          VR887    999     C. trachomatis                L1                  638     C. trachomatis                L2                  501     C. trachomatis                L3         VR903    821     C. psittaci           VR125    1.6     C. psittaci           VR629    0.9     C. psittaci           VR656    1.3     C. psittaci           VR813    1.2     ______________________________________      ##STR5##

                  TABLE 38     ______________________________________     HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9     TO ORGANISMS FOUND IN THE UROGENITAL TRACT                                  Ratio Counts     Organism            ATCC #   Bound*     ______________________________________     Achromobacter xylosoxidans                         27061    1.9     Acinetobacter lwoffii                         15309    1.2     Branhamella catarrhalis                         25238    1.2     Candida albicans    18804    2.4     Flavobacterium meningosepticum                         13253    1.1     Gardnerella vaginalis                         14018    1.3     Lactobacillus acidophilus                          4356    0.8     Listeria monocytogenes                         15313    0.7     Mycobacterium smegmatis                         14468    1.1     Moraxella osloensis 19976    1.3     Neisseria gonorrhoeae                         19424    2.3     Pasteurella multocida                          6529    1.0     Peptostreptococcus anaerobius                         27337    1.2     Streptococcus agalactiae                         13813    4.0     Streptococcus faecalis                         19433    2.6     ______________________________________      ##STR6##

                  TABLE 39     ______________________________________     HYBRIDIZATION OF CHLAMYDIA TRACHOMATIS PROBES 3-9     TO PHYLOGENETICALLY DIVERSE ORGANISMS                                Ratio Counts     Organism          ATCC #   Bound*     ______________________________________     Bacillus subtilis  6051    2.2     Bacteroides fragilis                       23745    1.6     Campylobacter jejuni                       33560    1.4     Chromabacterium violaceum                       29094    1.4     Deinococcus radiodurans                       35073    1.8     Derxia gummosa    15994    1.3     Enterobacter aerogenes                       13048    1.9     Escherichia coli  11775    1.9     Mycoplasma hominis                       14027    1.3     Pseudomonas cepacia                       17762    2.2     Proteus mirabilis 29906    2.2     Rahnella aquatilis                       33071    1.9     Rhodospirillum rubrum                       11170    1.9     Vibrio parahaemolyticus                       17802    2.0     Yersinia enterocolitica                        9610    2.5     ______________________________________      ##STR7##

EXAMPLE 12

Campylobacters are motile, microaerophilic, gram negative curved rods. The genus is quite diverse and distinct from other genera. Although the genus is well defined, some revision is occurring at the species level (Romaniuk, P. J. et al., J. Bacteriol. 169:2137-2141 (1987)). Three Campylobacter species, Campylobacter jejuni, C. coli and C. laridis, cause enteritis in humans. The disease includes diarrhea, fever, nausea, abdominal pain and in some cases, vomiting. These organisms cause an estimated 2 million infections per year in the United States (estimate based on the number of Salmonella and Shigella induced cases of diarrheal disease). Other members of the genus cause septicemias in humans and abortion and infertility in sheep and cattle.

Diagnosis of Campylobacter enteritis is currently dependent upon growth and isolation of the organism in culture, followed by a number of biochemical tests. Optimum growth of campylobacters requires special conditions such as low oxygen tension and high temperature (42° C.). No single set of conditions is recommended for isolation of all Campylobacter species.

The oligonucleotide sequences listed below, when used in a hybridization assay, hybridize to the 16S rRNA of the Campylobacter species of interest. The present invention has significant advantages over the prior art methods of detection of Campylobacter because one probe can detect all Campylobacters of interest; the other two probes detect the enteric Campylobacters and one can detect human isolates of Campylobacter. In addition, the probes have advantages over the prior art in terms of ease of the assay and greatly reduced time to identification and therefore, diagnosis.

The four probes which hybridize to the 16S rRNA of Campylobacter species of interest were constructed using three unique primers complementary to 16S rRNA. Sequences 1 and 2 were made using a 16S primer with the sequence 5'-GTA TTA CCG CGG CTG CTG GCA C-3'. Sequence 3 was made using a 16S primer with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. Sequence 4 was made with a 16S primer with the sequence 5'-GCT CGT TGC GGG ACT TAA CCC AAC AT-3'.

The following sequences were characterized and shown to hybridize to Campylobacter jejuni, C. coli and C. laridis. The phylogenetically nearest neighbors Vibrio parahaemolyticus and Wollinella succinogenes were used for comparison with the campylobacter sequences.

1. CGC TCC GAA AAG TGT CAT CCT CC

2. CCT TAG GTA CCG TCA GAA TTC TTC CC

3. GCC TTC GCA ATG GGT ATT CTT GGT G

4. GGT TCT TAG GAT ATC AAG CCC AGG

Sequence 1, from 16S rRNA, is 23 bases in length and has a Tm of 65° C. Sequence 2, from 16S rRNA, is 26 bases in length and has a Tm of 64° C. Sequence 3, from 16S rRNA, is 25 bases in length and has a Tm of 66° C. Sequence 4, from 16S rRNA, is 24 bases in length and has a Tm of 61° C. Sequence 1 is capable of hybridizing in the region corresponding to bases 405-428 of E. coli 16S rRNA; Sequence 2 is capable of hybridizing in the region corresponding to bases 440-475 of E. coli 16S rRNA; Sequence 3 is capable of hybridizing in the region corresponding to bases 705-735 of E. coli 16S rRNA; Sequence 4 is capable of hybridizing in the region corresponding to bases 980-1010 of E. coli 16S rRNA.

The reactivity and specificity of the probes for campylobacter was tested in hybridization assays. ³² P-end-labeled oligonucleotide probes were mixed with purified RNA or RNA released from cells in 0.1% sodium dodecyl sulfate. 0.5 ml of hybridization solution (41% diisobutyl sulfosuccinate, 30 mM sodium phosphate, pH 6.8, 0.7% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA) was added and the mixture incubated at 60° C. for 1 to 1.5 hour. Following incubation, 2 to 2.5 ml of separation solution (2% hydroxyapatite, 0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the mixture incubated at 60° C. for five minutes. The sample was centrifuged and the supernatant removed. 2.5 ml of wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the sample mixed, centrifuged and the supernatant removed. The radioactivity bound to the hydroxyapatite was determined by scintillation counting.

Table 40 indicates that the probes hybridize well to the campylobacter species of interest, C. jejuni, C. coli, and C. laridis. Probe 1 detects all of the Campylobacter species tested, probes 2 and 4 detect only the enteric campylobacters, and probe 3 detects all of the Campylobacter species except C. sputorum, an organism isolated from cattle. Thus all of the probes are useful for identifying campylobacter in stool samples. The choice of which probe to use for other applications would depend upon the level of specificity required (i.e., enteric campylobacters, or all Campylobacter species).

                  TABLE 40     ______________________________________     HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4     TO CAMPYLOBACTER SPECIES                     % Probe Bound (*)     Organism    ATCC#     1     2      3    4     ______________________________________     Campylobacter coli                 33559     64    70     52   49     C. fetus    27374     68    0.1    66   0.5     subsp. fetus     C. fetus    19438     66    0.7    54   1.2     subsp. venerealis     C. jejuni   33560     63    76     51   56     C. laridis  35221     74    73     64   52     C. sputorum 33562     71    3.0    2.5  0     subsp. bubulus     ______________________________________      (*) % Probe Bound = cpm bound to hybroxyapatite - cpm bound when no RNA      present/total cpm used in the assay

Table 41 shows that the probes do hot hybridize to closely related organisms or organisms found in the gastrointestinal tract.

                  TABLE 41     ______________________________________     HYBRIDIZATION OF CAMPYLOBACTER PROBES 1-4 TO     CLOSELY RELATED ORGANISMS AND ORGANISMS FOUND     IN THE GASTRO-INTESTINAL TRACT                       % Probe Bound (*)     Organism      ATCC#     1      2     3   4     ______________________________________     Bacteroides fragilis                   25285     0      0.2   0.7 0     Escherichia coli                   11775     1.3    0.5   0.5 0     Salmonella typhimurium                   14028     0      0     0.3 0     Shigella boydii                   29929     0      0.2   0.5 0     Shigella dysenteriae                   13313     0      0.7   0.2 0     Shigella flexneri                   29903     0      0     0.5 0     Shigella sonnei                   29930     0      0     0.1 0     Vibrio parahaemolyticus                   17802     0      1.9   0.1 0     Wollinella succinogenes                   29543     0.4    2.1   2.2 0     Yersinia pseudotuberculosis                   29833     0.6    0.2   1.7 0.3     ______________________________________      (*) % probe bound = cpm bound to hydroxyapatite - cpm bound when no RNA      present/total cpm used in the assay

The probes specific for the enteric Campylobacters, probes 2 and 4, were further tested and shown not to react with rRNAs of other organisms found in the gastrointestinal tract.

                  TABLE 42     ______________________________________     HYBRIDIZATION OF CAMPYLOBACTER PROBES 2 AND 4 TO     ORGANISMS FOUND IN THE GASTROINTESTINAL TRACT                         % Probe Bound (*)     Organism       ATCC#      Probe 2  Probe 4     ______________________________________     Citrobacter diversus                    27156      0        0     Clostridium perfringens                    13124      0        0     Enterobacter cloacae                    13047      0        0     Klebsiella pneumoniae                    23357      0        0.5     Proteus mirabilis                    25933      0        0     Serratia marcescens                    13880      0        0     Staphylococcus aureus                    e12600     Staphylococcus epidermidis                    14990      0        0.3     Streptococcus bovis                    33317      0        0     ______________________________________      (*) % probe bound = cpm bound to hydroxyapatite - cpm bound when no RNA      present/total cpm used in the assay

EXAMPLE 13

Streptococci are gram positive, oxidase negative coccoid bacteria. The genus has been divided into 18 groups, A-R, on the basis of group-specific carbohydrates. Group D streptococci are further subdivided into the enteroccocci (S. faecium, S. faecalis, S. avium and S. gallinarum and the non-enterococci S. bovis and S. equinus. S. faecium, S. faecalis and S. avium are considered the medically important enteroccocci. Some species of streptococcus are human pathogens; others are normal flora in the mouth and intestine but are capable of causing disease when introduced to other sites. Two examples are S. faecium and S. faecalis which are normally found in the intestine but may spread to cause bacteremia, wound infections, and as many as 10% of the urinary tract infections in the United States.

Current methods of detection of enterococci require culture of the specimen for 18-72 hours followed by a battery of biochemical tests. The oligonucleotide sequence shown below, when used in a hybridization assay, accurately detects Streptococcus faecalis, S. avium, and S. faecium. The inventive probe does not cross react with other Streptococci or Staphylococci which are very closely related in DNA homology. (Kiepper-Baez, 1981, 1982, Schliefer 1984.) The current invention also reduces the number of tests which must be run on a sample and greatly reduces the time to identification and thus, diagnosis. This represents a significant improvement over prior art methods.

The probe sequence was identified using a primer complementary to 16S rRNA with the sequence 5'-CCG CTT GTG CGG GCC CCC GTC AAT TC-3'. The following sequence was characterized and shown to be specific for three enterococci, S. faecium, S. faecalis and S. avium. The phylogenetically nearest neighbors S. agalactiae, S. bovis, S. pneumoniae and S. pyogenes were used for comparison with the sequences of interest.

1. TGC AGC ACT GAA GGG CGG AAA CCC TCC AAC ACT TA

The sequence is 35 bases in length and has a Tm of 72° C. It is capable of hybridizing in the region corresponding to bases 825-860 of E. coli 16S rRNA. To demonstrate the reactivity and specificity of the probe, it was used in a hybridization assay with purified RNA or RNA released from cells. A suspension containing at least 10⁷ cells in 2% sodium dodecyl sulfate was vortexed in the presence of glass beads. 0.1 ml of suspension was mixed with 0.1 ml of hybridization buffer (0.96 M sodium phosphate, pH 6.8, 0.002 M EDTA, 0.002 M EGTA) and incubated at 65° C. for 2 hours. After incubation, 5 ml of 2% hydoxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture was incubated at 65° C. for 10 minutes. The sample was centrifuged and the supernatant removed. Five ml of wash solution (0.12 M phosphate buffer, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the samples were vortexed, centrifuged, and the supernatant removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting. Table 43 shows that the probe reacts well with S. faeclum, S. faecalis, and S. avium, and does not react with other closely related organisms.

                  TABLE 43     ______________________________________     HYBRIDIZATION OF THE ENTEROCOCCUS PROBE     TO CLOSELY RELATED ORGANISMS     Organism          ATCC#   % Probe Bound     ______________________________________     Staphylococcus aureus                       12600   1.4     Streptococcus agalactiae                       13813   1.5     Streptococcus avium                       14025   22.7     Streptococcus bovis                       33317   1.4     Streptococcus faecalis                       19433   45.3     Streptococcus faecium                       19434   43.0     Streptococcus mitis                        9811   1.5     Streptococcus pneumoniae                        6306   1.5     Streptococcus pyogenes                       19615   1.3     ______________________________________

EXAMPLE 14

Pseudomonads are gram-negative, nonsporeforming, nonfermentative bacilli. Pseudomonads are common inhabitants of soil and water and rarely infect healthy individuals. When the organisms encounter already compromised patients, they can cause a variety of clinical syndromes including wound infections, post-surgical infections, septicemia, infant diarrhea and respiratory and urinary tract infections. Members of the genus Pseudomonas are particularly important to identify in a clinical sample because of the resistance of the organisms to antibiotics. Nucleic acid homology studies have divided the genus into five homology classes known as RNA groups I-V. Eighty-three percent of all clinical isolates of Pseudomonas are from RNA group I and Pseudomonas aeruginosa is by far the most common species isolated.

Current methods of detection of pseudomonas require culture of a patient sample for 24-72 hours, followed by a battery of biochemical tests. The oligonucleotide sequence below, when used in a hybridization assay, detects the clinically important group I pseudomonas. The present invention reduces the number of tests which must be run on a sample, and reduces the time to detection. This represents a significant improvement over prior art methods.

The sequence was obtained with a primer complementary to a conserved region on 23S rRNA with the sequence 5'-CTT TCC CTC ACG GTA-3'. The following sequence was shown to detect group I pseudomonads:

1. CAG ACA AAG TTT CTC GTG CTC CGT CCT ACT CGA TT

The probe is 35 bases in length and has a Tm of 70° C. It is capable of hybridizing to the RNA of group I Pseudomonas in the region corresponding to bases 365-405 of E. coli 23S rRNA. To demonstrate the reactivity and specificity of the probe, it was used in a hybridization assay. ³² P-end-labeled oligonucleotide was mixed with RNA released from at least 10⁷ organisms by standard methods in 0.48 M sodium phosphate pH 6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at 65° C. for two hours. After incubation, the RNA:DNA hybrids were bound to hydroxyapatite as described for previous examples and the radio-activity bound was determined by scintillation counting. Table 44 demonstrates that the probe reacted well with all 8 species of group I pseudomonads that were tested. The probe did not react with RNA from group II or group V organisms. A low reaction was seen with Pseudomonas acidovorans, a group III organism which represents <1% of all isolates of nonfermentative bacilli from clinical samples. Table 45 demonstrates that the probe does not react with other closely related organisms which were tested.

                  TABLE 44     ______________________________________     HYBRIDIZATION OF PSEUDOMONAS GROUP I     PROBE TO PSEUDOMONAS RNAs                                        % Probe*     Organism         Group    ATCC#    Bound     ______________________________________     Pseudomonas alcaligenes                      I        14909    24     Pseudomonas aeruginosa                      I        10145    83     Pseudomonas denitrificans                      I        13867    83     Pseudomonas fluorescens                      I        13525    82     Pseudomonas mendocina                      I        25411    79     Pseudomonas pseudoalcaligenes                      I        17440    78     Pseudomonas putida                      I        12633    80     Pseudomonas stutzeri                      I        17588    84     Pseudomonas cepacia                      II       25416    0     Pseudomonas pickettii                      II       27511    1.0     Pseudomonas acidovorans                      III      15668    11     Pseudomonas maltophilia                      V        13637    0.2     ______________________________________      *% Probe Bound = counts bound when RNA present - counts bound when no RNA      present/total counts used in the assay

                  TABLE 45     ______________________________________     HYBRIDIZATION OF PSEUDOMONAS GROUP I     PROBE TO RNAs OF CLOSELY RELATED ORGANISMS                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   1.6     Legionella pneumophila                        33155   0.6     Moraxella phenylpyruvica                        23333   0.3     Morganella morganii                        25830   0     Vibrio parahaemolyticus                        17802   0.6     ______________________________________      *% Probe Bound = counts bound when RNA present - counts bound when no RNA      present/total counts used in the assay

EXAMPLE 15

Examples 15-18 disclose probes for the Enterobacteriaceae, all of which are highly related at the DNA level. Even fewer differences exist at the rRNA level. For example, Proteus vulgaris 16S rRNA is 93% homologous to E. coli. These factors illustrate the difficulties associated with making rRNA probes specific for this group of organisms. Nevertheless, we have invented probes for Enterobacter cloacae, Proteus mirabilis, Salmonella and E. coli.

Members of the genus Enterobacter are motile, gram negative, non-sporeforming bacilli which belong in the family Enterobacteriaceae. The genus is a large and heterogeneous group. Eight species have been defined but only 5 are clinically significant. Enterobacter cloacae and E. aerogenes are the most common isolates and are associated with genitourinary, pulmonary, blood, central nervous system and soft tissue infections in humans.

The current method for identifying Enterobacter cloacae from patient samples involves culture of the specimen on agar plates for 18-24 hours, followed by a battery of biochemical tests. The oligonucleotide sequence described below, when used as a probe in a nucleic acid hybridization assay, accurately identifies Enterobacter cloacae. The present invention reduces the number of tests which must be run on a sample, the time to identification and therefore, diagnosis, and thus represents a significant improvement over prior art methods.

The probe specific for Enterobacter cloacae was obtained with a primer complementary to a conserved region of 23S rRNA with the sequence 5'-CAG TCA GGA GTA TTT AGC CTT-3'.

The following sequence was characterized and shown to be specific for E. cloacae. The phylogenetically nearest neighbors Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Salmonella enteritidis, and Citrobacter freundii were used as comparisons with the sequence of E. cloacae.

1. GTG TGT TTT CGT GTA CGG GAC TTT CAC CC

The probe is 29 bases in length and has a Tm of 68° C. It is capable of hybridizing to RNA of E. cloacae in the region corresponding to bases 305-340 of E. coli 23S rRNA. To demonstrate the reactivity and specificity of the probe for E. cloacae, it was used in a hybridization assay. ³² P-end-labeled oligonucleotide probe was mixed with RNA released from at least 10⁷ organisms in 1% sodium dodecyl sulfate, 0.48 M sodium phosphate, pH 6.8 (0.2 ml final volume) and incubated at 60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at 60° C. for 10 minutes. The sample was centrifuged and the supernatant removed. Five ml of wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added, the sample vortexed, centrifuged and the supernatant removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results are shown in Table 46 and demonstrates that the probe reacts well with E. cloacae and does not react with the RNA of closely related organisms.

                  TABLE 46     ______________________________________     HYBRIDIZATION OF ENTEROBACTER CLOACAE PROBE     TO CLOSELY RELATED ORGANISMS                               % Probe     Organisms Name    ATCC#   Bound     ______________________________________     Citrobacter freundii                        8090   1.8     Enterobacter aerogenes                       13048   1.4     Enterobacter cloacae                       13047   27.     Escherichia coli  11775   1.0     Klebsiella pneumoniae                       13883   1.7     Proteus mirabilis 29906   0.9     Proteus vulgaris  13315   0.6     Providencia stuartii                       29914   1.1     ______________________________________

Table 47 shows that the probe does not react with the RNA of organisms found in urine.

                  TABLE 47     ______________________________________     HYBRIDIZATION OF ENTEROBACTER CLOACAE     PROBE TO ORGANISMS FOUND IN URINE                                % Probe     Organisms Name     ATCC#   Bound     ______________________________________     Candida albicans   18804   0.8     Candida krusei     34135   0.8     Candida parapsilosis                        22019   0.9     Candida tropicalis  750    1.1     Pseudomonas aeruginosa                        10145   1.0     Serratia marcescens                        13880   1.6     Staphylococcus aureus                        12600   1.7     Staphylococcus epidermidis                        14990   1.4     Streptococcus agalactiae                        13813   2.5     Streptococcus faecium                        19434   1.5     Torulopsis glabrata                         2001   0.9     ______________________________________

EXAMPLE 16

Members of the genus Proteus are motile, gram negative, non-sporeforming bacilli which belong in the family Enterobacteriaceae. Four species of Proteus have been described and three of them, Proteus mirabilis, P. vulgaris, and P. penneri, cause human disease.

The most common type of proteus infection involves the urinary tract, but septicemia, pneumonia and wound infections also occur. Proteus mirabilis is the species most often isolated and may account for up to 10% of all acute, uncomplicated urinary tract infections. Species, rather than genus level identification of the causative organism is desirable because of differential antibiotic susceptibility among the species.

The current method for identifying Proteus mirabilis from patient samples involves culture of the specimen on agar plates for 18-24 hours, followed by a battery of biochemical tests. The oligonucleotide sequence described below, when used as a probe in a nucleic acid hybridization assay, accurately identifies Proteus mirabilis. The present invention reduces the number of tests which must be run on a sample, the time to identification and therefore, diagnosis and treatment. This represents a significant improvement over prior art methods.

The probe specific for Proteus mirabilis was obtained with a primer complementary to a conserved region of 23S rRNA with the sequence 5'-CAG TCA GGA GTA TTT AGC CTT-3'.

The following sequence was characterized and shown to be specific for P. mirabilis. The phylogenetically nearest neighbors Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Salmonella enteritidis were used as comparisons with the sequence of Proteus mirabilis.

1. CCG TTC TCC TGA CAC TGC TAT TGA TTA AGA CTC

This probe is capable of hybridizing to the RNA of P. mirabilis in the region corresponding to base 270-305 of E. coli 23S rRNA. The probe is 33 bases in length and has a Tm of 66° C. To demonstrate the reactivity and specificity of the probe for P. mirabilis, it was used in a hybridization assay. ³² P-end-labeled oligonucleotide probe was mixed with RNA released from at least 10⁷ organisms in 1% sodium dodecyl sulfate, 0.48 M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 ml final volume) and incubated at 64° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at 64° C. for 10 minutes. The sample was centrifuged and the supernatant removed. Five ml of wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added, the sample vortexed, centrifuged and the supernatant was removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results are shown in Table 48 and demonstrate that the probe reacts well with P. mirabilis and does not react with 27 other closely related bacteria. Table 49 shows that the probe does not react with 24 other phylogenetically diverse bacteria and two yeasts tested in the same manner as the organisms in Table 48.

                  TABLE 48     ______________________________________     HYBRIDIZATION OF PROTEUS MIRABILIS PROBE     TO CLOSELY RELATED ORGANISMS                                % Probe     Organism Name      ATCC#   Bound     ______________________________________     Citrobacter diversus                        27156   1.1     Citrobacter freundii                         8090   1.1     Citrobacter freundii                         6750   1.0     Enterobacter aerogenes                        13048   1.0     Enterobacter agglomerans                        27155   1.0     Enterobacter cloacae                        e13047  1.1     Enterobacter gergoviae                        33028   1.0     Enterobacter sakazakii                        29544   1.1     Escherichia coli   10798   1.2     Escherichia coli   11775   1.2     Escherichia coli   29417   1.2     Klebsiella oxytoca 13182   1.0     Klebsiella ozaenae 11296   1.1     Klebsiella planticola                        33531   0.9     Klebsiella pneumoniae                        13883   1.3     Klebsiella pneumoniae                        23357   1.1     Klebsiella rhinoscleromatis                        13884   1.2     Klebsiella terrigena                        33257   1.1     Klebsiella trevisanii                        33558   1.0     Kluyvera ascorbata 33433   0.9     Proteus mirabilis  25933   69.0     Proteus penneri    33519   2.5     Proteus vulgaris   13315   1.7     Providencia alcalifaciens                         9886   1.1     Providencia rettgeri                        29944   1.3     Providencia stuartii                        29914   1.1     Salmonella arizonae                        29933   1.1     Salmonella enteritidis                        13076   0.8     ______________________________________

                  TABLE 49     ______________________________________     HYBRIDIZATION OF PROTEUS MIRABILIS PROBE TO     PHYLOGENETICALLY DIVERSE ORGANISMS                                % Probe     Organsm Name       ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        33604   0.8     Bacillus subtilis   6051   1.2     Bacteroides fragilis                        23745   0.9     Branhamella catarrhalis                        25238   0.7     Campylobacter jejuni                        33560   1.0     Candida krusei     34135   0.8     Chromobacterium violaceum                        29094   1.1     Clostridium perfringens                        13124   0.9     Deinococcus radiodurans                        35073   0.8     Derxia gummosa     15994   0.8     Hafnia alvei       13337   0.9     Morganella morganii                        25830   0.9     Pseudomonas aeruginosa                        10145   1.0     Pseudomonas cepacia                        17762   0.9     Rahnella aquatilis 33071   0.9     Rhodospirillum rubrum                        11170   0.8     Serratia marcescens                        13880   0.9     Serratia odorifera 33077   0.9     Staphylococcus aureus                        e12600  0.8     Staphylococcus epidermidis                        14990   0.8     Streptococcus mitis                         9811   0.8     Streptococcus pneumoniae                        e6306   0.9     Torulopsis glabrata                         2001   0.9     Vibrio parahaemolyticus                        17802   0.8     Xanthomonas maltophilia                        13637   1.1     Yersinia enterocolitica                         9610   0.8     ______________________________________

EXAMPLE 17

Members of the genus Salmonella are motile, gram negative, non-sporeforming bacilli which belong in the family Enterobacteriaceae. All salmonellae are highly related and some microbiologists consider them to be one species. Five subgroups have been identified using nucleic acid homology studies and over 1400 different serotypes have been described. All serotypes have been implicated in human enteric disease ranging from self-limited gastroenteritis with mild symptoms, to severe gastroenteritis with bacteremia, to typhoid fever, a potentially life-threatening illness. S. cholerasuis, S. paratyphi A and S. typhi are the serotypes most often associated with severe disease and bacteremia. Diagnosis of Salmonella-induced enteritis is dependent upon detection of the organism in stool samples. Because infection occurs primarily by ingestion of contaminated milk, food and water, methods for identifying Salmonella in these products before release to consumers is critical.

Current methods for detection of members of the genus Salmonella involve culture of the specimen for 1-3 days on selective media followed by a battery of biochemical tests. Often an enrichment step is needed to isolate Salmonella from clinical samples or food products. The oligonucleotide sequences shown below, when used in a hydridization assay, accurately identify members of the genus Salmonella. The present inventive probes are specific for all members of the genus and do not react with the other closely related Enterobacteriaceae genera. These inventive probes reduce the number of tests which must be run on a sample and greatly reduce the time to identification. This represents a significant improvement over prior art methods.

The probes specific for the genus Salmonella were obtained with two primers complementary to 16S and 23S rRNA. Sequence 1 was obtained using a 16S primer with the sequence 5' TTA CTA GCG ATT CCG ACT TCA 3'. Sequence 2 was obtained using a 23S primer with the sequence 5' CAG TCA GGA GTA TTT AGC CTT 3'. The following sequences were characterized and shown to be specific for the genus Salmonella:

1. CTC CTT TGA GTT CCC GAC CTA ATC GCT GGC

2. CTC ATC GAG CTC ACA GCA CAT GCG CTT TTG TGT A

Sequence 1, from 16S rRNA, is 30 bases in length and has a Tm of 73° C. Sequence 2, from 23S rRNA, is 34 bases long and has a Tm of 71° C. These probes are capable of hybridizing in the regions corresponding to bases 1125-1155 of E. coli 16S rRNA and 335-375 of E. coli 23S rRNA, respectively. To demonstrate the reactivity and specificity of probe 1 for members of the genus Salmonella, ³² P-end-labeled oligonucleotide was tested as a probe in a hybridization reaction. Purified RNA, or RNA released from at least 10⁷ organisms by standard methods, was mixed with 1 ml hybridization buffer (final concentration 43% diisobutyl sulfosuccinate, 60 mM sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA) and incubated at 72° C. for 2-12 hours. Following incubation, 5 ml of separation solution (2% hydroxyapatite, 0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the sample were mixed, incubated at 72° C. for 5 minutes, centrifuged and the supernatants removed. Four ml of wash solution (0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate) was added and the samples were vortexed, centrifuged, and the supernatants removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting. The results shown in Table 50 indicate that a combination of the two probes hybridized to the 5 subgroups of Salmonella and to all 31 of the serotypes which were tested.

                  TABLE 50     ______________________________________     HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2     TO MEMBERS OF THE GENUS SALMONELLA                                      % Probe Bound     Subgroup             Organism         ATCC#   Probe 1                                            Probe 2     ______________________________________     I       Salmonella choleraesuis                              10708   24    40     I       Salmonella enteritidis                              13076   15    67     I       Salmonella paratyphi A                              9150    1.4   70     I       Salmonella sp. serotype                              9270    40    26             anatum     I       Salmonella sp. serotype                              12007   54    35             cubana     I       Salmonella sp. serotype give                              9268    12    40     I       Salmonella sp. serotype                              8326    53    33             heidelberg     I       Salmonella sp. serotype                              11646   36    46             illinois     I       Salmonella sp. serotype                              8387    35    32             montevideo     I       Salmonella sp. serotype                              29628   52    34             newington     I       Salmonella sp. serotype                              6962    3.4   36             newport     I       Salmonella sp. serotype                              15787   34    39             putten     I       Salmonella sp. serotype                              9712    28    30             saintpaul     I       Salmonella sp. serotype                              8400    38    43             senftenberg     I       Salmonella sp. serotype                              12004   29    29             simsbury     I       Salmonella sp. serotype                              15791   34    30             sloterdijk     I       Salmonella sp. serotype                              8391    32    41             thompson     I       Salmonella sp. serotype                              15611   35    2.6             vellore     I       Salmonella typhi 19430   7.0   21     I       Salmonella typhimurium                              14028   69    69     II      Salmonella salamae                              6959    3.0   46     II      Salmonella sp. serotype                              15793   6.6   30             maarssen     III     Salmonella arizonae                              33952   2.9   38     III     Salmonella arizonae                              12324   5.5   42     III     Salmonella arizonae                              29933   2.3   62     III     Salmonella arizonae                              29934   63    12     III     Salmonella arizonae                              12323   4.0   39     III     Salmonella arizonae                              12325   51    1.9     IV      Salmonella sp. serotype                              15783   5.8   8.0             harmelen     IV      Salmonella sp. serotype                              29932   7.5   40             ochsenzoll     V       Salmonella sp. serotype                              cdc1319 60    1.8             bongor     ______________________________________

The specificity of the probes for members of the genus Salmonella was demonstrated with hybridization reactions containing RNA from organisms closely related to Salmonella. The results are shown in Table 51.

                  TABLE 51     ______________________________________     HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2     TO RNA OF CLOSELY RELATED ORGANISMS                           % Probe Bound     Organism        ATCC#     Probe 1 Probe 2     ______________________________________     Citrobacter freundii                     6750      2.2     0     Edwardsiella tarda                     15947     0       0     Enterobacter agglomerans                     27155     0.6     0     Enterobacter cloacae                     13047     0       0     Enterobacter sakazakii                     29544     0       0     Escherichia coli                     10798     0       0     Escherichia coli                     29417     0       0     Klebsiella pneumoniae                     23357     0.7     0     Kluyvera ascorbata                     33433     0       0.5     Proteus mirabilis                     25933     0.2     0     Shigella flexneri                     29903     0       0     ______________________________________      *% Probe Bound = counts bound to hydroxyapatite  counts bound when no RNA      present/total counts used in assay

Table 52 shows that Salmonella probes 1 and 2 do not hybridize to phylogenetically diverse organisms.

                  TABLE 52     ______________________________________     HYBRIDIZATION OF SALMONELLA PROBES 1 AND 2 TO     RNA OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS                                % Probe Bound*     Organism         ATCC#     Probe 1 and Probe 2     ______________________________________     Acinetobacter calcoaceticus                      33604     1.1     0.1     Bacilius subtilis                      6051      0       0.5     Bacteroides fragilis                      23745     0.1     0     Branhamella catarrhalis                      25238     0.9     0     Campylobacter jejuni                      33560     0       0.2     Candida krusei   34135     0.4     0.3     Chromobacterium violaceum                      29094     1.7     0     Clostridium perfringens                      13124     0.3     0     Deinococcus radiodurans                      35073     1.6     0.1     Derxia gummosa   15994     1.2     0     Hafnia alvei     13337     1.8     0     Morganelli morganii                      25830     0       1.1     Pseudomonas aeruginosa                      10145     0.5     0.7     Pseudomonas cepacia                      17762     0       0     Pseudomonas maltophilia                      13637     1.9     0     Rahnella aquatilis                      33071     1.2     0.3     Rhodospirillum rubrum                      11170     0.9     0     Serratia marcescens                      13880     0       0     Serratia odorifera                      33077     2.6     0.2     Staphylococcus aureus                      e12600    0.2     0     Staphylococcus epidermidis                      14990     0       0     Streptococcus nitis                      9811      1.2     0.7     Streptococcus pneumoniae                      e6306     0       0     Torulopsis glabrata                      2001      0       0     Vibrio parahaemolyticus                      17802     0       0.2     Yersinia enterocolitica                      9610      0       0     ______________________________________      *% Probe Bound = Counts bound to hydroxyapatite  counts bound when no RNA      present/total counts used in assay

EXAMPLE 18

Escherichia coli is a gram negative, nonsporeforming bacillus which belongs in the family Enterobacteriaceae. Five species of Escherichia have been described: E. coli, which accounts for >99% of the clinical isolates, E. hermanii, E. blattae, E. vulneris and E. fergusonii. E. coli is a leading cause of urinary tract infections, bactermia and neonatal meningitidis, and can cause a type of gastroenteritis known as traveller's diarrhea.

The current method for identifying E. coli from patient samples involves culture of the specimen on agar plates for 18-72 hours, followed by a battery of biochemical tests on isolated colonies. The oligonucleotide sequence described below, when used as a probe in a nucleic acid hybridization assay, accurately detects E. coli even in the presence of other organisms. The present invention reduces the number of tests which must be run on a sample and reduces the time to identification and therefore diagnosis and treatment. This represents a significant improvement over prior art methods.

The probe specific for E. coli was derived from the published E. coli sequence (Brosius, et al. Proc. Natl. Acad. Sci. U.S.A. 75:4801-4805 (1978)), using Proteus vulgaris (Carbon, et al., Nuc. Acids Res. 9:2325-2333 (1981)), Klebsiella pneumoniae, Salmonella enteritidis, Enterobacter gergoviae and Citrobacter freundii for comparison. The probe sequence is shown below.

1. GCA CAT TCT CAT CTC TGA AAA CTT CCG TGG

It hybridizes to RNA of E. coli in the region of 995-1030 of 16s rRNA. The probe is 30 bases in length and has a T_(m) of 66° C. To demonstrate the reactivity and specificity of the probe for E. coli, it was used in a hybridization assay. ³² P-end-labeled oligonucleotide probe was mixed with two unlabeled oligonucleotides of sequences 5'-TGG ATG TCA AGA CCA GGT AAG GTT CTT CGC GTT GCA TCG-3' and 5'-CTG ACG ACA GCC ATG CAG CAC CTG TCT CAC GGT TCC CGA AGG CA-3' and with purified RNA, or RNA released from cells with detergent and heat, in 1% sodium dodecyl sulfate (SDS), 0.48 M sodium phosphate pH 6.8, 1 mM EDTA, 1 mM EGTA (0.2 ml final volume) and incubated at 60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at 60° C. for 10 minutes. The sample was centrifuged and the supernatant removed. Five ml of wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added, the sample vortexed, centrifuged and the supernatant was removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting.

An example of a use for this probe would be to detect E. coli in urine samples. Table 53 shows that the probe detects 7 out of 8 strains of E. coli tested. The probe also reacts with E. fergusonii, an organism which would only rarely be found in urine.

Table 54 shows that the probe does not react with any other genus tested except Shigella, another organism rarely isolated from urine. These results show that the probe will be useful in detecting E. coli from urine samples.

                  TABLE 53     ______________________________________     HYBRIDIZATION OF E. coli TO ESCHERICHIA SPECIES     Organism        ATCC#   % Probe Bound     ______________________________________     Escherichia coli                     10798   70     E. coli         11775   67     E. coli         23722   58     E. coli         25404   68     E. coli         25922   55     E. coli         29417   72     E. coli         33780   0.8     E. coli         35150   45     E. fergusonii   35469   55     E. hermanii     33650   0.7     E. vulneris     33821   0.8     ______________________________________

                  TABLE 54     ______________________________________     HYBRIDIZATION OF THE E. coli PROBE TO     CLOSELY RELATED ORGANISMS     Organism          ATCC#   % Probe Bound     ______________________________________     Citrobacter freundii                       6750    0.8     Citrobacter freundii                       8090    0.9     Citrobacter freundii                       29221   0.6     Citrobacter freundii                       33128   0.6     Enterobacter aerogenes                       13048   1.2     Enterobacter agglomerans                       27155   0.9     Enterobacter cloacae                       13047   0.9     Enterobacter gergoviae                       33023   0.7     Enterobacter sakazakii                       29544   0.6     Klebsiella oxytoca                       13182   0.7     Klebsiella pneumoniae                       13883   0.7     Proteus mirabilis 29906   0.7     Proteus vulgaris  13315   0.8     Shibella boydii   8700    76     Shigella dysenteriae                       13313   0.8     Shigella flexneri 29903   71     Shigella sonnei   29930   75     ______________________________________

EXAMPLE 19

The bacteria encompass a morphologically and physiologically diverse group of unicellular organisms which occupy most natural environments. Although many bacteria are harmless or beneficial to their environment or host, some are harmful and cause disease. The presence of any bacteria in some locations is undesirable or indicative of disease (e.g., culture media, pharmaceutical products, body fluids such as blood, urine or cerebrospinal fluid, and tissue biopsies). Low levels of bacteria are considered acceptable in other products such as drinking water and food products. Accordingly, there is a need for a means for detecting and quantitating bacteria in a sample.

The current method of detection and quantitation of total bacteria in a sample requires culture on multiple types of media under different conditions of temperature and atmosphere. To date, no single test exists to detect or quantitate all bacteria. The oligonucleotide sequences shown below, when used in a hybridization assay, detect a broad phylogenetic cross section of bacteria. The present invention reduces the number of tests which need to be performed and also reduces the time required for the assay. Comparison of the hybridization results from an unknown sample to a set of standards will allow some quantitation of the number of bacteria present. This represents a significant improvement over prior art methods.

The bacterial probes were designed following examination of published sequences of rRNA and sequences determined at Gen-Probe. The sequences used for the comparison include Agrobacterium tumefaciens (Yang et al., Proc. Natl. Acad. Sci. U.S.A., 82:4443, (1985), Anacystis nidulans (Tomioka and Sugiura. Mol. Gen. Genet. 191:46, (1983), Douglas and Doolittle Nuc. Acids Res. 12:3373, (1984), Bacillus subtilis (Green et al., Gene 37:261. (1985), Bacillus stearothermophilus (Kop et al., DNA 3:347, (1984), Bacteroides fragilis (Weisburg et al., J. Bacteriol. 164:230, (1985), Chlamydia psittaci (Weisburg et al., J. Bacteriol. 167:570. (1986)), Desulfovibrio desulfuricans (Oyaizu and Woese, System. Appl. Microbiol. 6:257, (1985); Escherichia coli, (Brosius et al., Proc. Natl. Acad. Sci, U.S.A. 77:201, (1980); Flavobacterium heparinum (Weisburg et al., J. Bacteriol. 164:230, (1985); Heliobacterium chlorum (Woese et al., Sience 229:762, (1985); Mycoplasma PG50 (Frydenberg and Christiansen, DNA 4:127, (1985); Proteus vulgaris (Carbon et al., Nuc. Acids Res. 9:2325, (1981); Pseudomonas testosteroni (Yang et al., Proc. Natl. Acad. Sci. U.S.A. 82:4443, (1985); Rcchalimaea quintana (Weisburg et al., Science 230:556, (1985); Saccharomyces cerevisiae (Rubstov et al., Nuc. Acids Res. 8:5779, (1980); Georgiev et al., Nuc. Acids Res. 9:6953, (1981); and human (Torczynski et al., DNA 4:283, (1985); Gonzalez et al., Proc. Natl. Acad. Sci. U.S.A. 82:7666, (1985)).

The following sequences were shown to hybridize to a broad phylogenetic cross section of bacteria and not to yeast or human rRNA:

1. CCA CTG CTG CCT CCC GTA GGA GTC TGG GCC

2. CCA GAT CTC TAC GCA TTT CAC CGC TAC ACG TGG

3. GCT CGT TGC GGG ACT TAA CCC AAC AT

4. GGG GTT CTT TTC GCC TTT CCC TCA CGG

5. GGC TGC TTC TAA GCC AAC ATC CTG

6. GGA CCG TTA TAG TTA CGG CCG CC

7. GGT CGG AAC TTA CCC GAC AAG GAA TTT CGC TAC C

Probe 1 is 30 bases long and has a Tm of 70° C. Probe 2 is 33 bases long and has a Tm of 69° C. Probe 3 is 26 bases long and has a Tm of 67° C. Probe 4 is 27 bases long and has a Tm of 69° C. Probe 5 is 24 bases long and has a Tm of 66° C. Probe 6 is 23 bases long and has a Tm of 62° C. Probe 7 is 34 bases long and has a Tm of 66° C. Probes 1-3 hybridize to 16S rRNA in the following regions, respectively, (corresponding to E. coli bases) 330-365; 675-715; and 1080-1110. Probes 4-7 hybridize to 23S rRNA in the following regions, respectively, (corresponding to E. coli bases) 460-490; 1050-1080; and 1900-1960 (probes 6 and 7). The oligonucleotides interact with regions on the rRNA which are highly conserved among eubacteria. This means that they can be used as bacterial probes in a hybridization assay. A second use is as a tool to obtain rRNA sequence. For example, an oligonucleotide can be hybridized to the rRNA of interest and extended with reverse transcriptase. The sequence of the resulting DNA can be determined and used to deduce the complementary rRNA sequence as described in the Detailed Description of the Invention.

One application of the invention is to detect bacteria in urine (bacteriuria). To demonstrate the reactivity and specificity of the probes for bacteria found in urine, they were used in hybridization assays. ³² P-end-labeled or ¹²⁵ I-labeled oligonucleotide probes were mixed with RNA released from cells by standard methods (e.g, the sonic disruption techniques described in Murphy et al., U.S. Pat. No. 5,374,522, detergent with glass beads, or enzymatic lysis). Probe was mixed with RNA in 0.48 M sodium phosphate, pH 6.8, 1 mM EDTA, 1 mM EGTA, 1% sodium dodecyl sulfate (0.2 ml final volume) and hybridized at 60° C. for 2 hours. Five ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the mixture incubated at 60° C. for 10 minutes. The mixture was centrifuged and the supernatant removed. Five ml of wash solution (0.12 M sodium phosphate, pH 6.8, 0.02% sodium dodecyl sulfate) was added and the sample was mixed, centrifuged and the supernatant removed. The amount of radioactivity bound to the hydroxyapatite was determined by scintillation counting. Tables 55-68 demonstrate the specificity of these probes and show that a combination of probes could be used to detect all bacteria which have been tested.

Table 55 shows that probe 1 hybridizes to the RNA of bacteria commonly isolated from urine and does not detect yeast RNA. Table 56 shows that probe 1 detects phylogenetically diverse bacteria and does not hybridize to human RNA.

                  TABLE 55     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 1     TO RNA OF ORGANISMS FOUND IN URINE                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   2.6     Candida krusei     34135   2.2     Candida parapsilosis                        22019   2.9     Candida tropicalis 750     2.5     Citrobacter freundii                        8090    69     Enterobacter aerogenes                        13048   70     Enterobacter cloacae                        13047   71     Escherichia coli   11775   67     Klebsiella oxytoca 13182   70     Klebsiella pneumoniae                        13883   72     Morganella morganii                        25830   66     Proteus mirabilis  29906   71     Proteus vulgaris   13315   67     Providencia stuartii                        29914   69     Pseudomonas aeruginosa                        10145   76     Pseudomonas fluorescens                        13525   73     Serratia marcescens                        13880   66     Staphylococcus aureus                        12600   57     Staphylococcus epidermidis                        14990   68     Streptococcus agalactiae                        13813   68     Streptococcus faecalis                        19433   51     Streptococcus faecium                        19434   53     Torulopsis glabrata                        2001    2.3     Ureaplasma urealyticum                        27618   54     ______________________________________

                  TABLE 56     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 1 TO RNAs     OF A PHYLOGENETIC CROSS SECTION OF ORGANISMS.                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   65     Bacillus subtilis  6051    73     Bacteroides fragilis                        23745   61     Branhamella catarrhalis                        25238   72     Campylobacter jejuni                        33560   64     Chlamydia trachomatis                        VR878   14     Chromabacterium violaceum                        29094   71     Clostridium perfringens                        13124   74     Corynebacterium xerosis                        373     38     Deinococcus radiodurans                        35073   47     Derxia gummosa     15994   65     Gardnerella vaginalis                        14018   67     Hafnia alvei       13337   60     Lactobacillus acidophilus                        4356    56     Moraxella osloensis                        19976   61     Mycobacterium smegmatis                        14468   47     Mycoplasma hominis 14027   58     Neisseria gonorrhoeae                        19424   58     Rahnella aquatilis 33071   74     Rhodospirillum rubrum                        11170   73     Vibrio parahaemolyticus                        17802   75     Human                      2.5     ______________________________________

Table 57 shows that Probe 2 hybridizes to the RNA of bacteria commonly found in urine except Ureaplasma urealyicum and does not hybridize to yeast rRNA.

                  TABLE 57     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 2     TO RNA OF ORGANISMS FOUND IN URINE                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   2.5     Candida krusei     34135   1.8     Candida parapsilosis                        22019   1.6     Candida tropicalis 750     1.4     Citrobacter freundii                        8090    61     Enterobacter aerogenes                        13048   57     Enterobacter cloacae                        13047   61     Escherichia coli   11775   67     Klebsiella oxytoca 13182   67     Klebsiella pneumoniae                        13883   51     Morganella morganii                        25830   69     Proteus mirabilis  29906   69     Proteus vulgaris   13315   69     Providencia stuartii                        29914   66     Pseudomonas aeruginosa                        10145   59     Pseudomonas fluorescens                        13525   58     Serratia marcescens                        13880   64     Staphylococcus aureus                        12600   60     Staphylococcus epidermidis                        14990   60     Streptococcus agalactiae                        13813   54     Streptococcus faecalis                        19433   37     Streptococcus faecium                        19434   58     Torulopsis glabrata                        2001    1.5     Ureaplasma urealyticum                        27618   3.2     ______________________________________

Table 58 shows that probe 2 detects phylogenetically diverse bacteria and does not hybridize to human rRNA.

                  TABLE 58     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 2 TO RNAs OF A CROSS     SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   76     Bacillus subtilis  6051    75     Bacteroides fragilis                        23745   2.0     Branhamella catarrhalis                        25238   70     Campylobacter jejuni                        33560   2.5     Chlamydia trachomatis                        VR878   16     Chromobacterium violaceum                        29094   61     Clostridium perfringens                        13124   66     Corynebacterium xerosis                        373     3.8     Deinococcus radiodurans                        35073   6.0     Derxia gummosa     15994   61     Gardnerella vaginalis                        14018   2.0     Hafnia alvei       13337   72     Lactobacillus acidophilus                        4356    50     Moraxella osloensis                        19976   64     Mycobacterium smegmatis                        14468   19     Mycoplasma hominis 14027   34     Neisseria gonorrhoeae                        19424   71     Rahnella aquatilis 33071   77     Rhodospirillum rubrum                        11170   1.5     Vibrio parahaemolyticus                        17802   73     Yersinia enterocolitica                        9610    76     Human                      2.0     ______________________________________

Table 59 shows that probe 3 hybridizes to the RNA of bacteria commonly found in urine and does not detect yeast rRNA.

                  TABLE 59     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNA OF     ORGANISMS FOUND IN URINE                                % Probe*     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   1.4     Candida krusei     34135   1.5     Candida parapsilosis                        22019   2.2     Candida tropicalis 750     2.6     Citrobacter freundii                        8090    79     Enterobacter aerogenes                        13048   40     Enterobacter cloacae                        13047   44     Escherichia coli   11775   67     Klebsiella oxytoca 13182   38     Klebsiella pneumoniae                        13883   45     Morganella morganii                        25830   57     Proteus mirabilis  29906   40     Proteus vulgaris   13315   51     Providencia stuartii                        29914   54     Pseudomonas aeruginosa                        10145   61     Pseudomonas fluorescens                        13525   56     Serratia marcescens                        13880   54     Staphylococcus aureus                        12600   37     Staphylococcus epidermidis                        14990   20     Streptococcus agalactiae                        13813   34     Streptococcus faecalis                        19433   20     Streptococcus faecium                        19434   47     Torulopsis glabrata                        2001    1.9     Ureaplasma urealyticum                        27618   26     ______________________________________

Table 60 shows that probe 3 detects phylogenetically diverse bacteria and does not hybridize to human rRNA.

                  TABLE 60     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 3 TO RNAs OF A CROSS     SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                                % Probe     Organism Name      ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   69     Bacillus subtilis  6051    35     Bacteroides fragilis                        23745   1.2     Branhamella catarrhalis                        25238   43     Campylobacter jejuni                        33560   55     Chlamydia trachomatis                        VR878   42     Chromobacterium violaceum                        29094   69     Clostridium perfringens                        13124   62     Corynebacterium xerosis                        373     23     Deinococcus radiodurans                        35073   30     Derxia gummosa     15994   67     Gardnerella vaginalis                        14018   40     Hafnia alvei       13337   56     Lactobacillus acidophilus                        4356    36     Moraxella osloensis                        19976   64     Mycobacterium smegmatis                        14468   77     Mycoplasma hominis 14027   1.5     Neisseria gonorrhoeae                        19424   26     Rahnella aquatilis 33071   66     Rhodospirillum rubrum                        11170   51     Vibrio parahaemolyticus                        17802   68     Yersinia enterocolitica                        9610    68     Human                      0.9     ______________________________________

Table 61 shows that probe 4 hybridizes to the RNA of bacteria commonly found in urine and does not detect yeast rRNA.

                  TABLE 61     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNA OF     ORGANISMS FOUND IN URINE                                % Probe     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   4.5     Candida krusei     34135   2.5     Candida parapsilosis                        22019   2.7     Candida tropicalis 750     2.5     Citrobacter freundii                        8090    55     Enterobacter aerogenes                        13048   52     Enterobacter cloacae                        13047   57     Escherichia coli   11775   70     Klebsiella oxytoca 13182   70     Klebsiella pneumoniae                        13883   43     Morganella morganii                        25830   74     Proteus mirabilis  29906   74     Proteus vulgaris   13315   73     Providencia stuartii                        29914   73     Pseudomonas aeruginosa                        10145   76     Pseudomonas fluorescens                        13525   79     Serratia marcescens                        13880   74     Staphylococcus aureus                        12600   73     Staphylococcus epidermidis                        14990   73     Streptococcus agalactiae                        13813   70     Streptococcus faecalis                        19433   37     Streptococcus faecium                        19434   63     Torulopsis glabrata                        2001    2.2     Ureaplasma urealyticum                        27618   43     ______________________________________

Table 62 shows that probe 4 detects phylogenetically diverse bacteria and does not hybridize to human rRNA.

                  TABLE 62     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 4 TO RNAs     OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE     ORGANISMS                                % Probe     Organism Name      ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   69     Bacillus subtilis  6051    55     Bacteroides fragilis                        23745   3.0     Branhamella catarrhalis                        25238   59     Campylobacter jejuni                        33560   65     Chlamydia trachomatis                        VR878   50     Chromobacterium violaceum                        29094   61     Clostridium perfringens                        13124   57     Corynebacterium xerosis                        373     9.5     Deinococcus radiodurans                        35073   63     Derxia gummosa     15994   65     Gardnerella vaginalis                        14018   57     Hafnia alvei       13337   67     Lactobacillus acidophilus                        4356    68     Moraxella osloensis                        19976   68     Mycobacterium smegmatis                        14468   28     Mycoplasma hominis 14027   74     Neisseria gonorrhoeae                        19424   76     Rahnella aquatilis 33071   68     Rhodospirillum rubrum                        11170   59     Vibrio parahaemolyticus                        17802   75     Yersinia enterocolitica                        9610    74     Human                      2.8     ______________________________________

Table 63 shows that probe 5 hybridizes to the RNA of bacteria commonly found in urine and does not detect yeast rRNA.

                  TABLE 63     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNA OF     ORGANISMS FOUND IN URINE                                % Probe     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   1.8     Candida krusei     34135   1.7     Candida parapsilosis                        22019   2.2     Candida tropicalis 750     1.8     Citrobacter freundii                        8090    39     Enterobacter aerogenes                        13048   38     Enterobacter cloacae                        13047   43     Escherichia coli   11775   31     Klebsiella oxytoca 13182   38     Klebsiella pneumoniae                        13883   66     Morganella morganii                        25830   50     Proteus mirabilis  29906   44     Proteus vulgaris   13315   52     Providencia stuartii                        29914   44     Pseudomonas aeruginosa                        10145   47     Pseudomonas fluorescens                        13525   25     Serratia marcescens                        13880   35     Staphylococcus aureus                        12600   26     Staphylococcus epidermidis                        14990   37     Streptococcus agalactiae                        13813   29     Streptococcus faecalis                        19433   14     Streptococcus faecium                        19434   33     Torulopsis glabrata                        2001    2.2     Ureaplasma urealyticum                        27618   73     ______________________________________

Table 64 shows that probe 5 detects phylogenetically divers bacteria and does not hybridize to human RNA.

                  TABLE 64     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNAs     OF A CROSS SECTION OF PHYLOGENETICALLY DIVERSE     ORGANISMS                                % Probe     Organism           ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   20     Bacillus subtilis  6051    53     Bacteroides fragilis                        23745   44     Branhamella catarrhalis                        25238   22     Campylobacter jejuni                        33560   35     Chromabacterium violaceum                        29094   59     Clostridium perfringens                        13124   63     Corynebacterium xerosis                        373     1.7     Deinococcus radiodurans                        35073   5.7     Derxia gummosa     15994   14     Gardnerella vaginalis                        14018   1.6     Hafnia alvei       13337   44     Lactobacillus acidophilus                        4356    1.5     Moraxella osloensis                        19976   7.2     Mycobacterium smegmatis                        14468   39     Mycoplasma hominis 14027   21     Neisseria gonorrhoeae                        19424   40     Rahnella aquatilis 33071   55     Rhodospirillum rubrum                        11170   17     Vibrio parahaemolyticus                        17802   66     Yersinia enterocolitica                        9610    64     Human                      1.6     ______________________________________

Table 65 shows that probe 6 hybridizes to the RNA of bacteria commonly found in urine and does not detect yeast rRNA.

                  TABLE 65     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 6 TO RNA OF     ORGANISMS FOUND IN URINE                                % Probe     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   3.0     Candida krusei     34135   2.0     Candida parapsilosis                        22019   2.2     Citrobacter freundii                        8090    54     Enterobacter aerogenes                        13048   50     Enterobacter cloacae                        13047   58     Escherichia coli   11775   63     Klebsiella oxytoca 13182   54     Klebsiella pneumoniae                        13883   55     Morganella morganii                        25830   60     Proteus mirabilis  29906   64     Proteus vulgaris   13315   67     Providencia stuartii                        29914   64     Pseudomonas aeruginosa                        10145   65     Pseudomonas fluorescens                        13525   31     Serratia marcescens                        13880   67     Staphylococcus aureus                        12600   53     Staphylococcus epidermidis                        14990   34     Streptococcus agalactiae                        13813   31     Streptococcus faecium                        19434   18     Torulopsis glabrata                        2001    2.5     ______________________________________

Table 66 shows that probe 6 detects some phylogenetically diverse bacteria and does not hybridize to human rRNA.

                  TABLE 66     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 5 TO RNAs     OF A CROSS SECTION OF PHYLOGENETICALLY     DIVERSE ORGANISMS                                % Probe     Organisms          ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   73     Bacteroides fragilis                        23745   7.0     Branhamella catarrhalis                        25238   4.0     Deinococcus radiodurans                        35073   5.5     Derxia gummosa     15994   3.0     Gardnerella vaginalis                        14018   2.0     Hafnia alvei       13337   3.5     Lactobacillus acidophilus                        4356    17     Moraxella osloensis                        19976   62     Mycoplasma hominis 14027   44     Rahnella aquatilis 33071   56     Yersinia enterocolitica                        9610    50     Human                      4.0     ______________________________________

Table 67 shows that probe 7 hybridizes to the RNA of bacteria commonly found in urine and does not detect yeast rRNA.

                  TABLE 67     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNA     OF ORGANISMS FOUND IN URINE                                % Probe     Organism           ATCC#   Bound     ______________________________________     Candida albicans   18804   2.1     Candida krusei     34135   2.0     Candida tropicalis 750     2.2     Citrobacter freundii                        8090    67     Enterobacter aerogenes                        13048   69     Enterobacter cloacae                        13047   78     Escherichia coli   11775   75     Klebsiella oxytoca 13882   79     Klebsiella pneumoniae                        13883   77     Morganella morganii                        25830   76     Proteus mirabilis  29906   77     Proteus vulgaris   13315   79     Providencia stuartii                        29914   64     Pseudomonas aeruginosa                        10145   76     Pseudomonas fluorescens                        13525   78     Serratia marcescens                        13880   66     Staphylococcus aureus                        12600   71     Staphylococcus epidermidis                        14990   75     Streptococcus agalactiae                        13813   70     Streptococcus faecalis                        19433   58     Streptococcus faecium                        19434   68     Torulopsis glabrata                        2001    2.4     Ureaplasma urealyticum                        27618   21     ______________________________________

Table 68 shows that probe 7 detects phylogenetically diverse bacteria and does not hybridize to human rRNA.

                  TABLE 68     ______________________________________     HYBRIDIZATION OF BACTERIAL PROBE 7 TO RNAs     OF A CROSS SECTION OF PHYLOGENETICALLY     DIVERSE ORGANISMS                                % Probe     Organism           ATCC#   Bound     ______________________________________     Acinetobacter calcoaceticus                        23055   86     Bacillus subtilis  6051    83     Bacteroides fragilis                        23745   69     Branhamella catarrhalis                        25238   74     Campylobacter jejuni                        33560   5.3     Chlamydia trachomatis                        VR878   41     Chromobacterium violaceum                        29094   69     Clostridium perfringens                        13124   68     Corynebacterium xerosis                        373     23     Deinococcus radiodurans                        35073   70     Derxia gummosa     15994   69     Gardnerella vaginalis                        14018   68     Hafnia alvei       13337   77     Moraxella osloensis                        19976   68     Mycobacterium smegmatis                        14468   64     Mycoplasma hominis 14027   4.0     Neisseria gonorrhoeae                        19424   53     Rahnella aquatilis 33071   72     Rhodospirillum rubrum                        11170   73     Vibrio parahaemolyticus                        17802   67     Yersinia enterocolitica                        9610    66     Human                      2.2     ______________________________________

EXAMPLE 20

Fungi encompass a morphologically and physiologically diverse group of simple eucaryotic organisms. We estimate, using published sequences of three fungi, Neurospora crassa, Podospora, and Saccharomyces, that the rRNA of fungi are 58-60% homologous to E. coli and 84-90% homologous to one another. Some fungi grow as single cells (yeasts), others as multinuclear filaments (molds) and still others can grow as either single cells or multicellular filaments (dimorphic fungi). Although many fungi are harmless inhabitants of their environments, others are harmful and cause disease. The presence of any fungi in some locations is undesirable or indicative of disease (e.g., culture media, pharmaceutical products, body fluids such as blood, urine or cerebrospinal fluid, and tissue biopsies). Low levels of fungi are considered acceptable in other products such as drinking water and food products. This has created the need for a means of detecting and quantitating fungi in a sample.

The current methods for detecting and quantifying fungi involve microscopic examination of samples and culture on different media. Although most yeasts can be grown from clinical samples in a matter of days, some filamentous fungi take up to four weeks culture time, after which special staining procedures, biochemical analysis and antigen tests are performed. The oligonucleotide sequences below, when used in a hybridization assay, detect the five yeasts most commonly isolated in the clinical setting, Candida albicans, Torulopsis glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei. Five other fungi representing the Trichosporon, Blastomyces, Cryptococcus and Saccharomyces genera are also detected. The present invention allows one step detection of these organisms and, in relation to culture, reduces the time to identification or elimination of these fungi as the cause of an infection. This represents a significant improvement over prior art methods.

The four probes which hybridize to the organisms of interest were identified using 3 primers complementary to conserved regions on 18S or 28S rRNA. Sequence 1 was obtained using an 18S primer with the sequence 5'-AGA ATT TCA CCT CTG-3'. Sequence 2 was obtained using a 28S primer with the sequence 5'-CCT TCT CCC GAA GTT ACG G-3'. Sequences 3 and 4 were obtained with a 28S primer with the sequence 5'-TTC CGA CTT CCA TGG CCA CCG TCC-3'. The following sequences were characterized and shown to hybridize to fungal rRNA. The sequences of Saccharomyces cerevisiae, Saccharomyces carlsberaensis, Escherichia coli and human rRNA were used for comparison with the sequences of interest.

1. CCC GAC CGT CCC TAT TAA TCA TTA CGA TGG

2. CGA CTT GGC ATG AAA ACT ATT CCT TCC TGT GG

3. GCT CTT CAT TCA ATT GTC CAC GTT CAA TTA AGC AAC AAG G

4. GCT CTG CAT TCA AAC GTC CGC GTT CAA TAA AGA AAC AGG G

Sequence 1, from 18S rRNA, is 30 bases in length and has a Tm of 68° C. Sequence 2, from 23S rRNA, is 32 bases in length and has a Tm of 67° C. Sequence 3, from 23S rRNA, is 40 bases in length and has a Tm of 66° C. Sequence 4, from 235 rRNA, is 40 bases in length and has a Tm of 68° C. Sequence 1 hybridizes in the region corresponding to position 845-880 of Saccharomyces cerevisiae 18S rRNA. Sequence 2 hybridizes in the region corresponding to position 1960-2000 of Saccharomyces cerevisiae 28S rRNA and sequences 3 and 4 hybridize in the region of 1225-1270 of the 28S rRNA.

To demonstrate the reactivity and specificity of these probes for fungal RNA, they were used in hybridization assays. ³² p- or ¹²⁵ I-labeled oligonucleotide probes were mixed with purified RNA or RNA released from cells by standard lysis techniques in 0.2 ml of 0.48 M sodium phosphate pH 6.8, 1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM EGTA and incubated at 60° C. for 2 hours. Following incubation, 5 ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added and the samples incubated 10 minutes at 60° C. The samples were centrifuged and the supernatants removed. Five ml of 0.12 M sodium phosphate pH 6.8, 0.02% sodium dodecyl sulfate was added, the samples were mixed, centrifuged and the supernatants removed. The results are shown in Table 69. Probe 1 detects all ten fungi which were tested, probe 2 detects all six of the yeasts which were tested, probe 3 detects five of the six yeasts, and probe 4 detects C. krusei only. Thus probe 4 could be used to detect and identify C. krusei in samples, probes 1, 2 or a combination of 3 and 4 could be used to detect the yeasts, and probe 1 could be used to detect any of the ten organisms listed in Table 69.

One potential use for these probes is to identify yeasts in urine samples or other normally sterile body fluids. The probes were hybridized to a panel of bacteria most commonly isolated from urine and shown not to react (Table 70). Table 71 shows that the probes do not hybridize to phylogenetically diverse bacteria or to human RNA.

                  TABLE 69     ______________________________________     HYBRIDIZATION OF YEAST PROBES TO YEAST RNA                             % Probe Bound     Organism      ATCC#     #1    #2     #3   #4     ______________________________________     Blastomyces dermatitidis                   C.I.      25    1.4    1.5  1.5     Candida albicans                   18804     40    63     56   2.0     C. krusei     34135     73    62     2.2  70     C. parapsilosis                   22019     71    63     65   2.0     C. tropicalis 750       62    71     71   2.0     Cryptococcus laurentii                   C.I.      43    1.4    1.5  1.5     Cryptococcus neoformans                   C.I.      60    1.3    1.5  1.6     Torulopsis glabrata                   2001      61    44     62   2.0     Trichosporon beigelii                   C.I.      57    1.3    2.1  1.5     Saccharomyces cerevisiae                   C.I.      41    67     53   1.9     ______________________________________      C.I. = Clinical isolute

                  TABLE 70     ______________________________________     HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNA     OF ORGANISMS FOUND IN URINE                               % Probe Bound     Organism        ATCC#     #1    #2    #3  #4     ______________________________________     Citrobacter freundii                     8090      1.5   1.7   1.5 2.1     Enterobacter aerogenes                     13048     2.5   1.9   2.0 2.0     Enterobacter cloacae                     13047     2.5   1.6   2.6 2.0     Escherichia coli                     11775     3.0   2.0   1.6 1.5     Klebsiella oxytoca                     13182     2.5   2.2   2.5 2.0     Klebsiella pneumoniae                     13883     2.5   2.2   2.1 2.0     Morganella morganii                     25830     2.0   2.8   1.7 1.9     Proteus mirabilis                     29906     2.5   1.9   2.3 2.0     Proteus vulgaris                     13315     2.0   2.2   2.0 1.5     Providencia stuartii                     29914     3.0   1.7   2.8 2.0     Pseudomonas aeruginosa                     10145     2.0   1.9   1.3 2.0     Pseudomonas fluorescens                     13525     2.5   2.7   2.1 2.0     Serratia marcescens                     13880     2.5   1.7   1.8 2.0     Staphylococcus aureus                     12600     2.0   1.7   1.8 2.0     Staphylococcus epidermidis                     14990     3.0   1.5   1.3 2.0     Streptococcus agalactiae                     13813     2.5   1.9   1.3 2.5     Streptococcus faecalis                     19433     1.7   3.3   3.5 1.9     Streptococcus faecium                     19434     2.0   2.9   2.1 1.5     Ureaplasma urealyticum                     27618     2.1   3.1   2.4 1.8     ______________________________________

                  TABLE 71     ______________________________________     HYBRIDIZATION OF FUNGAL PROBES 1-4 TO RNAs OF A CROSS     SECTION OF PHYLOGENETICALLY DIVERSE ORGANISMS                              % Probe Bound     Organism       ATCC#     #1    #2    #3  #4     ______________________________________     Acinetobacter calcoaceticus                    23055     2.5   2.5   2.0 1.9     Bacillus subtilis                    6051      2.0   2.8   2.4 2.4     Bacteroides fragilis                    23745     2.0   2.2   2.5 2.3     Branhamella catarrhalis                    25238     2.5   3.2   1.8 1.7     Campylobacter jejuni                    33560     2.5   2.1   2.0 1.9     Chlamydia trachomatis                    VR878     3.1   3.1   1.8 2.7     Chromobacterium violaceum                    29094     2.5   1.7   2.0 2.2     Clostridium perfringens                    13124     1.9   2.3   1.8 1.8     Corynebacterium xerosis                    373       1.6   4.8   1.8 1.1     Deinococcus radiodurans                    35073     2.0   1.6   2.1 0.8     Derxia gummosa 15994     3.0   1.5   1.7 1.8     Gardnerella vaginalis                    14018     2.0   2.2   1.3 1.2     Hafnia alvei   13337     1.0   2.5   1.7 1.6     Lactobacillus acidophilus                    4356      2.0   2.7   2.0 1.9     Moraxella osloensis                    19976     2.0   2.1   1.9 1.8     Mycobacterium smegmatis                    14468     1.6   1.8   1.8 1.7     Mycoplasma hominis                    14027     1.5   1.8   1.6 1.5     Neisseria gonorrhoeae                    19424     2.0   2.7   1.6 1.6     Rahnella aquatilis                    33071     2.0   2.7   2.3 2.1     Rhodospirillum rubrum                    11170     2.0   1.8   1.6 1.5     Vibrio parahaemolyticus                    17802     2.5   3.1   1.7 1.6     Yersinia enterocolitica                    9610      2.0   1.8   2.3 2.2     Human                    2.0   1.8   2.1 3.0     ______________________________________

Two derivatives of probe 1 also were made:

CCCGACCGTCCCTATTAATCATTACGATGGTCCTAGAAAC

CCCGACCGTCCCTATTAATCATTACGATGG

The first derivative works well at 65° C., the second at 60° C.

EXAMPLE 21

Gonorrhea is one of the most commonly reported bacterial infections in the United States, with over two million cases reported annually. This sexually transmitted disease usually results in anterior urethritis in males and involves the cervix in females. While severe complications and even sterility can occur in untreated individuals, asymptomatic infections are common, resulting in carriers who unknowingly spread the disease.

The causative agent, Neisseria gonorrhoeae, is a gram negative, oxidase positive diplococcus with stringent growth requirements. The method used for diagnosis depends on the site of infection and the patient symptoms. Gonococcal urethritis in males is diagnosed with good sensitivity and specificity using gram stain. Culture, requiring 24-72 hours, usually must be performed to confirm diagnosis of gonorrhea from all females and asymptomatic males. Following the detection of the organism from growth in culture, Neisseria gonorrhoeae must be identified by further tests such as carbohydrate degradation, coagglutination, fluorescent antibody screens or chromogenic enzyme substrate assays.

Neisseria gonorrhoeae is particularly difficult to detect and distinguish using a nucleic acid probe because it is very closely related to N. meningitidis. Data published in Kingsbury, D. T., J. Bacteriol. 94:870-874 (1967) shows a DNA:DNA homology for the two species of approximately 80-94%. Under guidelines established by the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics, Int'l J. System. Bacteriol. 37:463-464 (1987), the phylogenetic definition of a species generally means 70% or greater DNA:DNA homology. Despite the fact that these organisms may be considered to be the same species under established principles, we were able to make probes capable of distinguising them.

As expected, the rRNA homology between N. gonorrhoeae and N. meninaitidis is even greater because of known conserved regions. We noted a 1.0% difference between the 16S and a 1.1% difference between the 23S rRNA sequences of N. gonorrhoeae and N. meningitidis using our sequencing data.

Making a probe for N. gonorrhoeae was complicated by the fact that in some sites where N. meningitidis and N. gonorrhoeae differed, other Neisseria species were similar to N. gonorrhoeae. The few mismatches which exist between these two species are in the most variable regions, i.e., regions which vary not only between species, but also from strain to strain. Despite the fact that some believed the species could not be distinguished with nucleic acid probes at all, and others believed that rRNA was too conserved to be useful in probe diagnostics, we were able to make probes capable of differentiating N. gonorrhoeae and N. meningitidis.

The present invention has significant advantages over each of the prior art methods; the probes are more specific and much faster than culture methods. It also is believed that the probes are more sensitive, (i.e., able to detect a smaller number of organisms in a clinical sample) than prior art methods.

The primers used to identify these probe sequences had the following sequences:

1. GGCCGTTACCCCACCTACTAGCTAAT

2. GTATTACCGCGGCTGCTGGCAC

3. GCTCGTTGCGGGACTTAACCCACCAT

Each of the rRNA sites chosen to target had at least two mismatches to E. coli, N. meningitidis, N. cinerea, N. lactamica, N. mucosa, and Kingella kingae.

Oligonucleotides complementary to sequences adjacent to the probe regions were synthesized and used in the hydridization mix accoridng to Hogan et al., U.S. patent application Ser. No. 124,975, issued as U.S. Pat. No. 5,030,557, on Jul. 9, 1991, entitled "Means and Method for Enhancing Nucleic Acid Hybridization (the "helper" patent application).

The following sequences were characterized and shown to be specific for Neisseria gonorrhoeae. The phylogenetically nearest neighbors Neisseria meningitidis, N. lactamica, N. cinerea, N. mucosa, and Kingella kingae were used for comparison with the N. gonorrhoeae sequence.

1. CCG CCG CTA CCC GGT AC

2. TCA TCG GCC GCC GAT ATT GGC

3. GAG CAT TCC GCA CAT GTC AAA ACC AGG TA

Sequence 1, complementary to 16S rRNA in the region 125-150, is 17 bases in length and has a Tm of 56° C. Sequence 2, complementary to 16S rRNA in the region 455-485, is 21 bases in length and has a Tm of 63° C. Sequence 3, complementary to 16S rRNA in the region 980-1015, is 29 bases in length and has a Tm of 57° C.

The reactivity and specificity of the probes for Neisseria gonorrhoeae was demonstrated with a hybridization assay. The three oligonucleotide probes were iodinated and mixed with unlabeled oligonucleotides of sequence 5'-CCC CTG CTT TCC CTC TCT AGA CGT ATG CGG TAT TAG CTG ATC TTT CG-3', 5'-GCC TTT TCT TCC CTG ACA AAA GTC CTT TAC AAC CCG-3', 5'-GGC ACG TAG TTA GCC GGT GCT TAT TCT TCA GGT AC-3', and 5'-GGT TCT TCG CGT TGC ATC GAA TTA ATC CAC ATC ATC CAC CGC-3', and with purified RNA in 0.48 M sodium phosphate, pH 6.8, 0.5% sodium dodecyl sulfate (SDS) and incubated at 60° C. for one hour. Following incubation, 4 ml of 2% hydroxyapatite, 0.12 M sodium phosphate pH 6.8, 0.02% SDS was added and the mixture was incubated at 60° C. for 5 minutes. The samples were centrifuged and the supernatants were removed. Five ml of wash solution (0.12 M sodium phosphate pH 6.8, 2% SDS) was added and the samples were mixed, centrifuged, and the supernatants removed. The amount of radioactivity bound to the hydroxyapatite was determined in a gamma counter.

Table 72 shows that the probes hybridize well to N. gonorrhoeae RNA and do not hybridize to the other species tested.

                  TABLE 72     ______________________________________     HYBRIDIZATION OF NEISSERIA GONORRHOEAE     PROBES 1-3 TO NEISSERIA AND KINGELLA RNAS     Organisms         ATCC#   % Probe Bound     ______________________________________     Kingella kingae   23332   0.09     Neisseria cinerea 14685   0.04     N. gonorrhoeae    19424   48.4     N. lactamica      23970   0.07     N. meningitidis serogroup A                       13077   0.04     N. meningitidis serogroup B                       13090   0.04     N. meningitidis serogroup C                       13102   0.04     N. mucosa         19696   0.07     N. subflava       14799   0.05     ______________________________________

    GAG GAT TCC GCA CAT GTC AAA ACC AGG     GAG GAT TCC GCA CAT GTC AAA ACC AGG TAA     CCC GCT ACC CGG TAC GTT C     CCG CTA CCC GGT ACG TTC.

Although the above examples of performance were determined using the standard assay format previously described, the specific probes may be used under a wide variety of experimental conditions. For example, additives may be included to the reaction solutions to provide optimal reaction conditions for accelerated hybridization. Such additives may include buffers, chelators, organic compounds and nucleic acid precipitating agents such as detergents, dihydroxybenzene, sodium dodecyl sulfate, sodium diisobutyl sulfosuccinate, sodium tetradecyl sulfate, sarkosyl and the alkali metal salts and ammonium salts of SO₄ ²⁻, PO₄ ³⁻, Cl⁻ and HCOO⁻. Such additives can be utilized by one skilled in the art to provide optimal conditions for the hybridization reaction to take place. These conditions for accelerated hybridization of single stranded nucleic acid molecules into double stranded molecules are the subject of the above-noted U.S. Pat. No. 5,132,207.

The present invention can be carried out on nonviral organisms from purified samples or unpurified clinical samples such as sputum, feces, tissue, blood, spinal or synovial fluids serum, urine or other bodily fluids, or other samples such as environmental or food samples. Prior to cell breakage and hybridization, the cells can be suspended or placed in solution. In the case of the unpurified samples referred to above, the cells may remain intact and untreated in their own biological environment prior to the assay.

The probes of the present invention may be used in an assay either alone or in combination with different probes. Several individual probes also can be linked together during nucleic acid synthesis. This results in one probe molecule which contains multiple probe sequences, and therefore, multiple specificities. For example, a single nucleic acid molecule can be synthesized which contains both the Mycobacterium avium and the Mycobacterium intracellulare sequences described in Examples 1 and 2. When hybridized with either M. avium or M. intracellulare rRNA this probe will hybridize completely. If the two probe sequences were combined separately in an assay only one half of the mixed individual probes will hybridize with either M. avium or M. intracellulare rRNA. Other embodiments also may be practiced within the scope of the claims. For example, probes may be labelled using a variety of labels, as described within, and may be incorporated into diagnostic kits. 

We claim:
 1. A probe comprising an oligonucleotide 15 to 100 nucleotides in length able to hybridize to an Enterobacter cloacae nucleic acid target region to form a detectable target:probe duplex under high stringency hybridization assay conditions, said target region corresponding to, or fully complementary to and of the same length as a nucleic acid corresponding to, bases 305-340 of E. coli 23S rRNA; wherein said oligonucleotide comprises a segment of 15 contiguous bases which is at least 75% complementary to a target sequence of 15 contiguous nucleotides present in said target region and said oligonucleotide does not hybridize to nucleic acid from Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Citrobacter freundii to form a detectable non-target:probe duplex under said hybridization conditions.
 2. The probe of claim 1, wherein said target region corresponds to bases 305-340 of E. coli 23S rRNA.
 3. The probe of claim 1, wherein said 15 contiguous base region is present in a nucleic acid sequence selected from the group consisting of:' GTGTGTTTTCGTGTACGGGACTTTCACCC, and the sequence fully complementary and of the same length thereto.
 4. The probe of any of claims 1-3, wherein said oligonucleotide comprises a segment of 15 contiguous bases which is at least 90% complementary to said target sequence of 15 contiguous nucleotides.
 5. The probe of claim 4, wherein said oligonucleotide comprises a segment of 15 contiguous bases which is 100% complementary to said target sequence of 15 contiguous nucleotides.
 6. The probe of claim 5, wherein said high stringency hybridization assay conditions comprise 0.12M phosphate buffer containing equimolar amounts of Na₂ HPO₄ and NaH₂ PO₄, 1 mM EDTA and 0.02% sodium dodecyl sulfate at 65° C.
 7. The probe of claim 4, wherein said oligonucleotide is 15-50 bases in length.
 8. A method for determining whether Enterobacter cloacae may be present in a sample comprising the steps of:a) providing to said sample a probe comprising an oligonucleotide able to hybridize to an Enterobacter cloacae nucleic acid target region to form a detectable target:probe duplex under hybridization assay conditions, said target region corresponding to, or fully complementary to and of the same length as a nucleic acid corresponding to, bases 305-340 of E. coli 23S rRNA; wherein said oligonucleotide comprises a segment of 10 contiguous bases which is at least 75% complementary to a target sequence of 10 contiguous nucleotides present in said target region and said oligonucleotide does not hybridize to nucleic acid from Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Citrobacter freundii to form a detectable non-target:probe duplex under said hybridization conditions, and b) detecting hybridization of said oligonucleotide to nucleic acid present in said sample under said hybridization conditions as an indication that Enterobacter cloacae may be present.
 9. The method of claim 8, wherein said oligonucleotide comprises a sequence selected from the group consisting of:5' GTGTGTTTTCGTGTACGGGACTTTCACCC, and the sequence of the same length and fully complementary thereto.
 10. The method of claim 8, wherein said target region corresponds to bases 305-340 of E. coli 23S rRNA.
 11. The method of claim 8, wherein said target sequence of 10 contiguous nucleotides is present in a nucleic acid sequence selected from the group consisting of:5' GTGTGTTTTCGTGTACGGGACTTTCACCC, and the sequence fully complementary and of the same length thereto.
 12. The method of any of claims 8, 10 and 11, wherein said oligonucleotide comprises a segment of 10 contiguous bases which is at least 90% complementary to said target sequence of 10 contiguous nucleotides.
 13. The method of claim 12, wherein said oligonucleotide comprises a segment of 10 contiguous bases which is 100% complementary to said target sequence of 10 contiguous nucleotides.
 14. The method of claim 12, wherein said oligonucleotide is 15-50 bases in length.
 15. The method of claim 12, wherein said probe further comprises either a detectable isotopic label or a detectable non-isotopic label.
 16. The method of claim 15, wherein said probe comprises a detectable non-isotopic label selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.
 17. The method of claim 16, wherein said probe comprises an acridinium ester label. 